Novel Group of STAT3 Pathway Inhibitors and Cancer Stem Cell Pathway Inhibitors

ABSTRACT

The present invention relates to the use of a novel class of cancer stem cell pathway (CSCP) inhibitors; to methods of using such compounds to treat refractory, recurrent, or metastatic cancers; to methods of selective killing cancer cells by using such corn pounds with specific administration regimen; to methods of targeting cancer stem cells by inhibiting Stat3 pathway; to methods of using novel compounds in the treatment of conditions or disorders in a mammal related to aberrant Stat3 pathway activity; and to processes for preparing such compounds and intermediates thereof and to the pharmaceutical compositions of relevant compounds, and to the specific methods of administration of these compounds.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/591,960, filed on Oct. 3, 2019, which is a continuation of U.S.patent application Ser. No. 16/236,948, filed on Dec. 31, 2018, which isa continuation of U.S. patent application Ser. No. 15/655,366, filed onJul. 20, 2017, which is a division of U.S. patent application Ser. No.12/677,513, filed on Mar. 20, 2012, which is a is a national stageapplication filed under 35 U.S.C. § 371 from International ApplicationSerial No. PCT/US2008/075903, filed on Sep. 10, 2008, and published asWO 2009/036099 on Mar. 19, 2009, which claims the benefit of priority toU.S. Provisional Application Ser. No. 60/971,144, filed on Sep. 10, 2007and U.S. Provisional Application Ser. No. 61/013,372, filed on Dec. 13,2007. The contents of the above applications are incorporated herein byreference in their entireties.

FIELD OF THE INVENTION

The present invention relates generally to the use of Stat3 pathwayinhibitors to treat conditions. More specifically, the invention relatesto the use of Stat3 pathway inhibitors to target cancer stem cells andto treat other disorders. Even more specifically, the invention relatesto the use of naphtho [2,3-b]furan-4,9-dione and related compounds toinhibit Stat3, to target cancer stem cells, and to treat malignantdiseases. The invention also relates to treatment for refractory,recurrent, or metastatic cancers, and to processes for preparingrelevant compounds and intermediates thereof, and to pharmaceuticalcomposition of relevant compounds.

BACKGROUND OF THE INVENTION

Cancer Stem cells (CSCs).

In recent years, a new model of tumorigenesis has gained wideacceptance, where it is hypothesized that only a small fraction of theentire tumor mass are responsible for the tumorigenic activities withinthe tumor, whereas the old or clonal genetic model posits that all themutated tumor cells contribute equally to such tumorigenic activities.This small fraction of tumorigenic cells, according to the new model,are transformed cells with stem-cell-like qualities and are called“cancer stem cells” (CSCs). Bonnet and Dick first demonstrated, in vivo,the presence of CSCs in acute myeloid leukemia (AML) during the 1990s.Their data showed that only a small subpopulation of human AML cells hadthe ability to transfer AML when transplanted into immunodeficient micewhile other AML cells were incapable of inducing leukemia. Later, theseCSCs were shown to have the same cellular markers, CD34⁺/CD38⁻, asprimitive hematopoietic stem cells [1]. Since then, researchers havefound CSCs conclusively in various types of tumors including those ofthe brain, breast, skin, prostate, and so on.

The CSC model of tumorigenesis would explain why tens or hundreds ofthousands of tumor cells need to be injected into an experimental animalin order to establish a tumor transplant. In human AML, the frequency ofthese cells is less than 1 in 10,000 [2]. Even though rare within agiven tumor cell population, there is mounting evidence that such cellsexist in almost all tumor types. However, as cancer cell lines areselected from a sub-population of cancer cells that are specificallyadapted to grow in tissue culture, the biological and functionalproperties of cancer cell lines can undergo dramatic changes. Therefore,not all cancer cell lines contain CSCs.

Cancer stem cells share many similar traits with normal stem cells. Forexample, CSCs have self-renewal capacity, namely, the ability to giverise to additional tumorigenic cancer stem cells, typically at a slowerrate than other dividing tumor cells, as opposed to a limited number ofdivisions. CSCs also have the ability to differentiate into multiplecell types, which would explain histological evidence that not only manytumors contain multiple cell types native to the host organ, but alsothat heterogeneity is commonly retained in tumor metastases. CSCs havebeen demonstrated to be fundamentally responsible for tumorigenesis,cancer metastasis, and cancer reoccurrence. CSCs are also called tumorinitiating cells, cancer stem-like cells, stem-like cancer cells, highlytumorigenic cells, tumor stem cells, solid tumor stem cells, or supermalignant cells.

The existence of cancer stem cells has fundamental implications forfuture cancer treatments and therapies. These implications aremanifested in disease identification, selective drug targeting,prevention of cancer metastasis and recurrence, and development of newstrategies in fighting cancer.

The efficacy of current cancer treatments are, in the initial stages oftesting, often measured by the size of the tumor shrinkage, i.e., theamount of tumor mass that is killed off. As CSCs would form a very smallproportion of the tumor and have markedly different biologiccharacteristics than their more differentiated progenies, themeasurement of tumor mass may not necessarily select for drugs that actspecifically on the stem cells. In fact, cancer stem cells appear to beresistant to radiotherapy (XRT) and also refractory to chemotherapeuticand targeted drugs [3-5]. Normal somatic stem cells are naturallyresistant to chemotherapeutic agents—they have various pumps (such asMDR) that pump out drugs, and DNA repair proteins. Further, they alsohave a slow rate of cell turnover while chemotherapeutic agents targetrapidly replicating cells. Cancer stem cells, being the mutatedcounterparts of normal stem cells, may also have similar mechanisms thatallow them to survive drug therapies and radiation treatment. In otherwords, conventional chemotherapies and radiotherapies killdifferentiated or differentiating cells, which form the bulk of thetumor that are unable to generate new highly tumorigenic cancer stemcells. The population of cancer stem cells that gave rise to thedifferentiated and differentiating cells, on the other hand, couldremain untouched and cause a relapse of the disease. A further dangerfor conventional anti-cancer therapy is the possibility thatchemotherapeutic treatment leaves only chemotherapy-resistant cancerstem cells, and the ensuing recurrent tumor will likely also beresistant to chemotherapy.

Since the surviving cancer stem cells can repopulate the tumor and causerelapse, it is imperative that anti-cancer therapies include strategiesagainst CSCs (see FIG. 1). This is akin to eliminating the roots inorder to prevent dandelions from regrowth even if the weed's groundlevel mass has been cut [6]. By selectively targeting cancer stem cells,it becomes possible to treat patients with aggressive, non-resectabletumors and refractory or recurrent cancers, as well as preventing thetumor metastasis and recurrence. Development of specific therapiestargeting cancer stem cells may improve survival and the quality of lifeof cancer patients, especially for sufferers of metastatic cancers. Thekey to unlocking this untapped potential is the identification andvalidation of pathways that are selectively important for cancer stemcell self-renewal and survival. Unfortunately, though multiple pathwaysunderlying tumorigenesis in cancer or self-renewal in embryonic andadult stem cells have been elucidated in the past, no pathways have beenidentified and validated for cancer stem cell self-renewal and survival.

There has also been a lot of research into the identification andisolation of cancer stem cells. Methods used mainly exploit the abilityof CSCs to efflux drugs, or are based on the expression of surfacemarkers associated with cancer stem cells.

For example, since CSCs are resistant to many chemotherapeutic agents,it is not surprising that CSCs almost ubiquitously overexpress drugefflux pumps such as ABCG2 (BCRP-1) [7-11], and other ATP bindingcassette (ABC) superfamily members [12, 13]. Accordingly, the sidepopulation (SP) technique, originally used to enrich hematopoetic andleukemic stem cells, was also employed to identify and isolate CSCs[14]. This technique, first described by Goodell et al., takes advantageof differential ABC transporter-dependent efflux of fluorescent dyessuch as Hoechst 33342 to define and isolate a cell population enrichedin CSCs [10, 15]. Specifically, the SP is revealed by blocking drugefflux with verapamil, at which point the dyes can no longer be pumpedout of the SP.

Researchers have also focused on finding specific markers thatdistinguish cancer stem cells from the bulk of the tumor. Most commonlyexpressed surface markers by the cancer stem cells include CD44, CD 133,and CD 166 [16-22]. Sorting tumor cells based primarily upon thedifferential expression of these surface marker(s) have accounted forthe majority of the highly tumorigenic CSCs described to date.Therefore, these surface markers are well validated for identificationand isolation of cancer stem cells from the cancer cell lines and fromthe bulk of tumor tissues.

Stat3 Pathway.

There are many different genetic defects in mammalian or human cancercells, and many have been studied in the quest to cure cancer. Forexample, the p53 tumor suppressor has been found to be defective oraltogether absent in more than half of the human cancers. The STAT(Signal Transducers and Activator of Transcription) protein family arelatent transcription factors activated in response to cytokines/growthfactors to promote proliferation, survival, and other biologicalprocesses. Among them, Stat3 is activated by phosphorylation of acritical tyrosine residue mediated by growth factor receptor tyrosinekinases, Janus kinases, or the Src family kinases, etc. These kinasesinclude, but are not limited to EGFR, JAKs, Abl, KDR, c-Met, Src, andHer2 [23]. Upon tyrosine phosphorylation, Stat3 forms homo-dimers,translocates to the nucleus, binds to specific DNA-response elements inthe promoter regions of the target genes, and induces gene expression[24].

In normal cells, Stat3 activation is transient and tightly regulated,lasting from 30 minutes to several hours. However, Stat3 is found to beaberrantly active in a wide variety of human cancers, including all themajor carcinomas as well as some hematologic tumors. Stat3 playsmultiple roles in cancer progression. As a potent transcriptionregulator, it targets genes involved in many important cellularfunctions, such as Bcl-xl, c-Myc, cyclin D1, Vegf, MMP-2, and survivin[25-30]. It is also a key negative regulator of tumor immunesurveillance and immune cell recruitment [31-33].

Ablating Stat3 signaling by antisense, siRNA, a dominant-negative formof Stat3, and/or blockade of tyrosine kinases inhibits certain cancercell lines or tumors in vitro and/or in vivo [24, 26, 34, 35]. But noclear link between Stat3 and cancer stem cell functionality has everbeen empirically made. Nor have researchers found an effective Stat3pathway inhibitor to explore potential therapeutic uses with regard tocancers that have been found to contain cancer stem cells. As describedearlier, cancer stem cells (CSCs) have been recently demonstrated to befundamentally responsible for tumorigenesis, metastasis, andreoccurrence, and should be taken into consideration in designing anycurative therapy that targets a tumor known to have these cells nomatter how small a fraction of the tumor mass they may constitute.

In diseases other than cancer, over-activation of Stat3 by Interleukin 6(IL6) has been demonstrated in a number of autoimmune and inflammatorydiseases [36]. Recently, it has been revealed that the Stat3 pathwayalso promotes pathologic immune responses through its essential role ingenerating TH17 T cell responses [37]. In addition, IL6-Stat3 pathwaymediated inflammation has been found to be the common causative originfor atherosclerosis, peripheral vascular disease, coronary arterydisease, hypertension, osteroprorosis, type 2 diabetes, and dementia.

SUMMARY

The present invention is predicated, in part, on empirical evidenceprovided herein that Stat3 plays a key role in both the survival andself-renewal capacity of cancer stem cells (CSCs) across a broadspectrum of cancers. Accordingly, a first aspect of the invention isdirected to a method of inhibiting a cancer stem cell where the methodcomprises inhibiting at least some, most, or substantially all (e.g.,30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%), of the Stat3 pathwayactivity in the cancer stem cell through a Stat3 pathway inhibitor. Themethod inhibits the CSC from self-renewal or kills the CSC. The methodcan be carried out in vitro, or in vivo to treat a cancer, especiallycancers that have CSCs and have aberrant, e.g., overactive Stat3 pathwayactivities. These two criteria can be met by virtue of institutionalknowledge, i.e., the patient's cancer is of a type known to have CSCsand aberrant Stat3 pathway activities, or can be confirmed from theindividual patient, e.g., through tests conducted on a biopsy. In apreferred embodiment, the CSCs are known or otherwise confirmed to haveaberrant Stat3 pathway activities.

Cancers that are currently known to have both CSCs and aberrant Stat3pathway activities include and are not limited to: breast cancer, headand neck cancer, lung cancer, ovarian cancer, pancreatic cancer,colorectal carcinoma, prostate cancer, melanoma, sarcoma, liver cancer,brain tumors, multiple myeloma, and leukemia. Many of the metastaticforms of these cancers have also been found to have both CSCs andaberrant Stat3 pathway activities, such as metastatic breast cancer. Inone feature, methods of the present invention can be practiced to treata cancer selected from this group. In one embodiment, methods of theinvention can be practiced to treat a cancer selected from thefollowing: lung cancer, breast cancer, cervical cancer, colorectalcarcinoma, liver cancer, head and neck cancer, pancreatic cancer,gastric cancer, and prostate cancer.

Further, as CSCs have been demonstrated to be fundamentally responsiblefor tumorigenesis, cancer metastasis and cancer reoccurrence, methods ofthe invention can be practiced to treat cancer that is metastatic,refractory to a chemotherapy or radiotherapy, inherently resistant tochemotherapy or has relapsed in the subject after an initial treatment.In one embodiment, the Stat3 pathway inhibitor is isolated, purified orsynthetic, and can be selected from the group consisting of a smallmolecule Stat3 inhibitor, an RNAi agent against Stat3, an antisenseagent against Stat3, a peptidomimetic Stat3 inhibitor, and a G-quartetoligodeoxynucleotide Stat3 inhibitor. The mechanism of inhibition can beselected from the group consisting of substantially inhibitingphosphorylation of the Stat3 protein, substantially inhibitingdimerization of the Stat3 protein, substantially inhibiting nucleartranslocation of the Stat3 protein, substantially inhibiting DNA-bindingactivity of the Stat3 protein, and substantially inhibitingtranscription activities of the Stat3 protein.

In one embodiment, the inhibitor is a compound selected from the groupconsisting of 2-(1-hydroxyethyl)-naphtho[2,3-b]furan-4,9-dione,2-acetyl-7-chloro-naphtho[2,3-b]furan-4,9-dione,2-acetyl-7-fluoro-naphtho[2,3-b]furan-4,9-dione,2-acetylnaphtho[2,3-b]furan-4,9-dione,2-ethyl-naphtho[2,3-b]furan-4,9-dione, phosphoric acidmono-[1-(4,9-dioxo-3a,4,9,9a-tetrahydro-naphtho[2,3-b]furan-2-yl)-vinyl]ester,phosphoric acid1-(4,9-dioxo-3a,4,9,9a-tetrahydro-naphtho[2,3-b]furan-2-yl)-vinyl esterdimethyl ester, an enantiomer, diastereomer, tautomer, and a salt orsolvate thereof (hereafter referred to as the “Compound of theInvention”).

In a second aspect, the present invention provides a method ofinhibiting cellular Stat3 pathway activity in a cell. The methodincludes administering to the cell an effective amount of the Compoundof the Invention such that at least unwanted Stat3 pathway activity inthe cell is reduced, for example, by at least 20%, 30%, 40%, 50%, 60%,70%, 80%, 90% or 95%, In one embodiment, the cell is a CSC, or otherwisecancerous. The method may induce cell death or inhibit self-renewal inthe cell. The method can be carried out in vitro or in vivo.

In a third aspect, the present invention provides a method of treatingor preventing a disorder associated with aberrant Stat3 pathway activityin a subject, the method comprising administering to the subject atherapeutically effective amount of a pharmaceutical compositioncomprising the Compound of the Invention such that at least aberrantStat3 pathway activity is reduced. In one feature, aberrant Stat3pathway activity can be identified by expression of phosphorylated Stat3or a surrogate upstream or downstream regulator of Stat3phosphorylation. The disorder can be a cancer. In one embodiment, thecancer is known to have aberrant Stat3 pathway activities, and includebut are not limited to: breast cancer, head and neck cancer, lungcancer, ovarian cancer, pancreatic cancer, colorectal carcinoma,prostate cancer, renal cell carcinoma, melanoma, hepatocellularcarcinomas, cervical cancer, sarcomas, brain tumors, gastric cancers,multiple myeloma, leukemia, and lymphomas. The disorder may also be anon-cancerous condition known to be associated with aberrant Stat3pathway activity, and in one embodiment, is selected from the groupconsisting of an autoimmune disease, an inflammatory disease,inflammatory bowel diseases, arthritis, autoimmune demyelinationdisorder, Alzheimer's disease, stroke, ischemia reperfusion injury, andmultiple sclerosis.

In a fourth aspect, the present invention provides a method of treatinga patient and includes the steps of identifying a patient by aberrantStat3 pathway activity and administering to the patient atherapeutically effective amount of the Compound of the Invention. Inone embodiment, the step of identifying the patient by aberrant Stat3pathway activity comprises testing expression of phosphorylated Stat3 orof a surrogate upstream or downstream regulator of Stat3phosphorylation. The step of identifying the patient by aberrant Stat3pathway activity may comprise testing diseased tissue or fluid takenfrom the patient, which may be part of a tumor.

In a fifth aspect, the present invention provides a method of treating apatient and includes the steps of identifying a patient diagnosed with adisorder associated with aberrant Stat3 pathway activity andadministering to the patient a therapeutically effective amount of theCompound of the Invention. In one embodiment, the step of identifyingthe patient comprises testing for at least one biomarker that indicatesthe disorder in the patient.

In a sixth aspect, the present invention provides a kit that includes atleast one agent for diagnosing a disorder associated with aberrant Stat3pathway activity, which can be testing for a biomarker that indicatesthe presence of the disorder, and a therapeutically effective amount ofthe Compound of the Invention.

In a seventh aspect, the present invention provides a kit that includesat least one agent for diagnosing aberrant Stat3 pathway activity, and atherapeutically effective amount of the Compound of the Invention. Inone embodiment, the agent tests the expression of phosphorylated Stat3or of a surrogate upstream or downstream regulator of Stat3phosphorylation.

In an eighth aspect, the present invention provides a method ofinhibiting one or more cancer stem cells. The method includesadministering to the cancer stem cell an effective amount of theCompound of the Invention. The method can be carried out in vitro or invivo to treat a cancer in a subject. In one embodiment, the cancer isknown to have CSCs, and include but are not limited to: breast cancer,head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer,colorectal carcinoma, prostate cancer, liver cancer, melanoma, multiplemyeloma, brain tumors, sarcomas, medulloblastoma, and leukemia. In oneembodiment, the cancer is metastatic. In another embodiment, the canceris refractory to a chemotherapy or radiotherapy. For example, the cancercan be inherently resistant to chemotherapy. In yet another embodiment,the cancer has relapsed in the subject after an initial treatment.

In a ninth aspect, the present invention provides a method ofidentifying a drug candidate capable of inhibiting a cancer stem cell,the method comprising screening for a drug candidate that inhibits Stat3pathway activity. The drug candidate, in one embodiment, is capable ofinducing cell death in the cancer stem cell, and in another embodiment,of inhibiting self-renewal of the CSC. In various embodiments, the drugcandidate is a small molecule Stat3 inhibitor, an RNAi agent againstStat3, an antisense agent against Stat3, a peptidomimetic Stat3inhibitor, or a G-quartet oligodeoxynucleotides Stat3 inhibitor. Thedrug candidate may have the capacity selected from the following:substantially inhibiting phosphorylation of the Stat3 protein,substantially inhibiting dimerization of the Stat3 protein,substantially inhibiting nuclear translocation of the Stat3 protein,substantially inhibiting DNA-binding activity of the Stat3 protein, andsubstantially inhibiting transcription activities of the Stat3 protein.

In a tenth aspect, the present invention provides a method of treating asubject for cancer refractory to a standard treatment, the methodcomprising administering to the subject a therapeutically effectiveamount of a pharmaceutical composition comprising the Compound of theInvention. The standard treatment can be, for example, a chemotherapy, aradiotherapy, and/or surgery. In one embodiment, the cancer isinherently resistant to chemotherapy.

In an eleventh aspect, the present invention provides a method oftreating or preventing cancer relapse in a subject, the methodcomprising administering to the subject a therapeutically effectiveamount of a pharmaceutical composition comprising the Compound of theInvention. In one embodiment, the pharmaceutical composition isadministered as an adjuvant therapy after surgery.

In a twelfth aspect, the present invention provides a method of treatingor preventing cancer metastasis in a subject, the method comprisingadministering to the subject a therapeutically effective amount of apharmaceutical composition comprising the Compound of the Invention. Inone embodiment, the pharmaceutical composition is administered as anadjuvant therapy after surgery.

In a thirteenth aspect, the present invention provides a method ofselectively targeting cancer cells in a subject, e.g., for treating amalignant disease, the method comprising administering to a subject apharmaceutical composition comprising the Compound of the Invention,such that the compound concentration in the subject's plasma is notmaintained above a critical concentration for more than 24 hour aftereach dose, thereby selectively killing cancer cells while substantiallysparing normal cells. Alternatively, according to the inventive method,the compound plasma concentration is not above the criticalconcentration at a certain time point after each dose, e.g., 12, 16, 20or 24 hours. In one embodiment, the pharmaceutical composition isadministered such that the compound concentration in the subject'splasma is not maintained above a critical concentration, e.g.,continuously, for more than a duration selected from the groupconsisting of 12, 16, and 20 hours after each dose. In variousembodiments, the critical concentration is about 100 μM, about 50 μM,about 30 μM, or about 20 μM. In one embodiment, the cancer cells arepart of a cancer selected from the group, or any of its subgroup,consisting of liver cancer, head and neck cancer, pancreatic cancer,gastric cancer, renal cancer, sarcoma, multiple myeloma, metastaticbreast cancer, leukemia, lymphoma, esophageal cancer, brain tumor,glioma, bladder cancer, endometrial cancer, thyroid cancer, bile ductcancer, bone cancer, eye cancer (retinoblastoma), gallbladder cancer,pituitary cancer, rectal cancer, salivary gland cancer, nasal pharyngealcancer, breast cancer, lung cancer, colon cancer, prostate cancer,ovarian cancer, neuroblastoma, cervix cancer, leukemia, melanoma, oralepithermoid, keratinocyte, and skin cancer. In another embodiment, thecancer cells are part of a cancer selected from the group consisting oflung cancer, breast cancer (including the metastatic kind), cervicalcancer, colorectal carcinoma, liver cancer, head and neck cancer,pancreatic cancer, gastric cancer, and prostate cancer.

In a fourteenth aspect, the present invention provides a method oftreating cancer in a subject, the method comprising administering to thesubject a therapeutically effective amount of a pharmaceuticalcomposition comprising the Compound of the Invention. Methods accordingto this aspect of the invention can be applied to treat cancer similarto those described with regard to previous aspects of the invention. Inone feature, the subject of the treatment is a mammal, e.g., a human.

In a fifteenth aspect, the present invention provides a pharmaceuticalcomposition that comprises the Compound of the Invention, i.e., acompound selected from the group consisting of2-(1-hydroxyethyl)-naphtho[2,3-b]furan-4,9-dione,2-acetyl-7-chloro-naphtho[2,3-b]furan-4,9-dione,2-acetyl-7-fluoro-naphtho[2,3-b]furan-4,9-dione,2-acetylnaphtho[2,3-b]furan-4,9-dione,2-ethyl-naphtho[2,3-b]furan-4,9-dione, phosphoric acidmono-[1-(4,9-dioxo-3a,4,9,9a-tetrahydro-naphtho[2,3-b]furan-2-yl)-vinyl]ester, phosphoric acid1-(4,9-dioxo-3a,4,9,9a-tetrahydro-naphtho[2,3-b]furan-2-yl)-vinyl esterdimethyl ester, and a pharmaceutically acceptable salt or solvatethereof, and a pharmaceutically-acceptable excipient, carrier, ordiluent. In one feature, the composition is suitable for oral, nasal,topical, rectal, vaginal or parenteral administration, or intravenous,subcutaneous or intramuscular injection.

In a sixteenth aspect, the present invention also provides a process ofpreparing some of the Compounds of the Invention. The method prepares acompound of formula 4-6,

wherein R₁ is H, Cl, or F, by reacting a compound of formula 4-4,

with a ketone in a solvent while in the presence of a base and anoxidizing agent. The oxidizing agent can be, for example, 02, Bra orCBrCl₃. In one embodiment, the reaction is carried out in an open aircontainer. In one feature, all the steps of the process take place inone pot, i.e., in the same container. In various exemplary embodiments,the solvent may be tetrahydrofuran (THF), dioxane, or toluene, and thebase may be 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), triethyl amine, ordiisopropylethyl amine.

In an embodiment of the above process, the ketone is a compound offormula 4-3.

The above process can further include the following steps:

reacting a compound of formula 4-1,

with bromide with or without solvent to produce a compound of formula4-2,

and subsequently reacting the compound of formula 4-2 in a solvent inthe presence of a base to produce the compound of formula 4-3.

In a seventeenth aspect, the present invention provides a compound ofthe formula phosphoric acidmono-[1-(4,9-dioxo-3a,4,9,9a-tetrahydro-naphtho[2,3-b]furan-2-yl)-vinyl]ester.

In an eighteenth aspect, the present invention provides a compound ofthe formula phosphoric acid1-(4,9-dioxo-3a,4,9,9a-tetrahydro-naphtho[2,3-b]furan-2-yl)-vinyl esterdimethyl ester.

In a nineteenth aspect, the present invention provides a process ofpreparing the compound phosphoric acidmono-[1-(4,9-dioxo-3a,4,9,9a-tetrahydro-naphtho[2,3-b]furan-2-yl)-vinyl]ester,the process comprising reacting compound2-acetylnaphtho[2,3-b]furan-4,9-dione with a solution selected from thegroup consisting of lithium bis(trimethylsilyl)amide, sodiumbis(trimethylsilyl)amide, and potassium bis(trimethylsilyl)amid,followed with adding a solution of dimethyl chlorophosphate. The processmay further comprise purifying a crude product obtained from thereaction by dissolving the product in CH₂Cl₂, washing it with saturatedNH₄CL and water, drying it over MgSO₂, and subsequently running theproduct through column chromatography.

In a twentieth aspect, the present invention provides a process ofpreparing the compound phosphoric acid1-(4,9-dioxo-3a,4,9,9a-tetrahydro-naphtho[2,3-b]furan-2-yl)-vinyl esterdimethyl ester, the process comprising reacting compound phosphoric acidmono-[1-(4,9-dioxo-3a,4,9,9a-tetrahydro-naphtho[2,3-b]furan-2-yl)-vinyl]esterwith trimethylsilyl bromide. The process can further comprise purifyinga crude product obtained from the reaction by a semi-prep-HPLC.

Other aspects and embodiments of the present invention are set forth orwill be readily apparent from the following detailed description of theinvention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the differences between cancer-stem-cell-specific andconventional cancer therapies.

FIG. 2 shows the Stat3 pathway in cancer.

FIG. 3A shows that Stat3 is constitutively active in Hoechst SidePopulation cells.

FIG. 3B shows that Stat3 is constitutively active in CD133⁺ cells.

FIG. 4A and FIG. 4B show that Stat3 knockdown in cancer stem cellsinduces apoptosis.

FIG. 5 shows that Stat3 knockdown in cancer stem cells inhibits cancerstem cell spherogenesis.

FIG. 6 shows that compound 401 inhibits Stat3 transcription activationactivity.

FIG. 7A shows that compound 401 inhibits Stat3 DNA-binding activity innuclear extract.

FIG. 7B shows that compounds 401, 416 and 418 inhibit Stat3 DNA-bindingactivity in nuclear extract.

FIG. 8A shows the sorting and analysis of the Hoechst Side Population.

FIG. 8B shows that Hoechst Side Population is as sensitive as non-sidepopulation to compound 401.

FIG. 9A shows that compound 401 is apoptotic to Hoechst Side Populationcells.

FIG. 9B shows that compound 401 is apoptotic to CD133⁺ cells.

FIG. 10A and FIG. 10B show that compound 401 blocks CD44^(high) sphereformation.

FIG. 11 shows that compound 401 induces apoptosis in cancer cells.

DETAILED DESCRIPTION

As used herein, the singular form “a”, “an”, and “the” include pluralreferences unless the context clearly dictate otherwise. For example,the term “a cell” includes a plurality of cells including mixturesthereof.

The terms “isolated” or “purified” as used herein refer to a materialthat is substantially or essentially free from components that normallyaccompany it in its native state. Purity and homogeneity are typicallydetermined using analytical chemistry techniques such as polyacrylamidegel electrophoresis or high performance liquid chromatography.

As used herein, the terms “cancer stem cell(s)” and “CSC(s)” areinterchangeable. CSCs are mammalian, and in preferred embodiments, theseCSCs are of human origin, but they are not intended to be limitedthereto. Cancer stem cells are defined and functionally characterized asa population of cells originating from a solid tumor that: (1) haveextensive proliferative capacity; 2) are capable of asymmetric celldivision to generate one or more kinds of differentiated progeny withreduced proliferative or developmental potential; and (3) are capable ofsymmetric cell divisions for self-renewal or self-maintenance. Othercommon approaches to characterize CSCs involve morphology andexamination of cell surface markers, transcriptional profile, and drugresponse. CSCs are also called in the research literature tumor/cancerinitiating cells, cancer stem-like cells, stem-like cancer cells, highlytumorigenic cells, tumor stem cells, solid tumor stem cells, drugsurvival cells (DSC), drug resistant cells (DRCs) or super malignantcells.

As used herein, the term “self-renewal” refers to cancer stem cells'ability to give rise to new tumorigenic cancer stem cells to replenishor increase their number.

As used herein, the terms “cancer” and “cancerous” refer to or describethe physiological condition in mammals in which a population of cellsare characterized by unregulated cell growth. “Cancer cells” and “tumorcells” as used herein refer to the total population of cells derivedfrom a tumor including both non-tumorigenic cells, which comprise thebulk of the tumor cell population, and tumorigenic stem cells (cancerstem cells). Examples of cancer include, but are not limited to,carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particularexamples of such cancers include squamous cell cancer, small-cell lungcancer, non-small cell lung cancer, adenocarcinoma of the lung, squamouscarcinoma of the lung, cancer of the peritoneum, hepatocellular cancer,gastrointestinal cancer, pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney cancer, liver cancer,prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma andvarious types of head and neck cancer.

“Tumor” as used herein refers to any mass of tissue that result fromexcessive cell growth or proliferation, either benign (noncancerous) ormalignant (cancerous) including pre-cancerous lesions.

“Metastasis” as used herein refers to the process by which a cancerspreads or transfers from the site of origin to other regions of thebody with the development of a similar cancerous lesion at the newlocation. A “metastatic” or “metastasizing” cell is one that losesadhesive contacts with neighboring cells and migrates via thebloodstream or lymph from the primary site of disease to invadeneighboring body structures.

As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to humans, non-human primates,rodents, and the like, which is to be the recipient of a particulartreatment. Typically, the terms “subject” and “patient” are usedinterchangeably herein in reference to a human subject.

Terms such as “treating” or “treatment” or “to treat” or “alleviating”or “to alleviate” as used herein refer to both 1) therapeutic measuresthat cure, slow down, lessen symptoms of, and/or halt progression of adiagnosed pathologic condition or disorder and 2) prophylactic orpreventative measures that prevent or slow the development of a targetedpathologic condition or disorder. Thus those in need of treatmentinclude those already with the disorder; those prone to have thedisorder; and those in whom the disorder is to be prevented. A subjectis successfully “treated” according to the methods of the presentinvention if the patient shows one or more of the following: a reductionin the number of or complete absence of cancer cells; a reduction in thetumor size; inhibition of or an absence of cancer cell infiltration intoperipheral organs including the spread of cancer into soft tissue andbone; inhibition of or an absence of tumor metastasis; inhibition or anabsence of tumor growth; relief of one or more symptoms associated withthe specific cancer; reduced morbidity and mortality; and improvement inquality of life.

As used herein, the term “inhibiting”, “to inhibit” and theirgrammatical equivalents, when used in the context of a bioactivity,refer to a down-regulation of the bioactivity, which may reduce oreliminate the targeted function, such as the production of a protein orthe phosphorylation of a molecule. In particular embodiments, inhibitionmay refers to a reduction of about 20%, 30%, 40%, 50%, 60%, 70%, 80%,90% or 95% of the targeted activity. When used in the context of adisorder or disease, the terms refer to success at preventing the onsetof symptoms, alleviating symptoms, or eliminating the disease, conditionor disorder.

The term “pharmaceutically-acceptable excipient, carrier, or diluent” asused herein means a pharmaceutically-acceptable material, composition orvehicle, such as a liquid or solid filler, diluent, excipient, solventor encapsulating material, involved in carrying or transporting thesubject pharmaceutical agent from one organ, or portion of the body, toanother organ, or portion of the body. Each carrier must be “acceptable”in the sense of being compatible with the other ingredients of theformulation and not injurious to the patient. Some examples of materialswhich can serve as pharmaceutically-acceptable carriers include: sugars,such as lactose, glucose and sucrose; starches, such as corn starch andpotato starch; cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; powderedtragacanth; malt; gelatin; talc; excipients, such as cocoa butter andsuppository waxes; oils, such as peanut oil, cottonseed oil, saffloweroil, sesame oil, olive oil, corn oil and soybean oil; glycols, such aspropylene glycol; polyols, such as glycerin, sorbitol, mannitol andpolyethylene glycol; esters, such as ethyl oleate and ethyl laurate;agar; buffering agents, such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol; phosphate buffer solutions; and other non-toxiccompatible substances employed in pharmaceutical formulations. Wettingagents, emulsifiers and lubricants, such as sodium lauryl sulfate,magnesium stearate, and polyethylene oxide-polypropylene oxide copolymeras well as coloring agents, release agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants can alsobe present in the compositions.

The compounds of the present invention may form salts which are alsowithin the scope of this invention. Reference to a compound of thepresent invention herein is understood to include reference to saltsthereof, unless otherwise indicated. The term “salt(s)”, as employedherein, denotes acidic and/or basic salts formed with inorganic and/ororganic acids and bases. In addition, when a compound of the presentinvention contains both a basic moiety, such as but not limited to apyridine or imidazole, and an acidic moiety such as but not limited to acarboxylic acid, zwitterions (“inner salts”) may be formed and areincluded within the term “salt(s)” as used herein. Pharmaceuticallyacceptable (i.e., non-toxic, physiologically acceptable) salts arepreferred, although other salts are also useful, e.g., in isolation orpurification steps which may be employed during preparation. Salts ofthe compounds of the present invention may be formed, for example, byreacting a compound I, II or III with an amount of acid or base, such asan equivalent amount, in a medium such as one in which the saltprecipitates or in an aqueous medium followed by lyophilization.

Solvates of the compounds of the invention are also contemplated herein.Solvates of the compounds of the present invention include, for example,hydrates.

Recent studies have uncovered the presence of cancer stem cells (CSCs)with an exclusive ability to regenerate tumors. These CSCs exist inalmost all tumor types and are functionally linked with continuedmalignant growth, cancer metastasis, recurrence, and cancer drugresistance. CSCs and their more differentiated progenies appear to havemarkedly different biologic characteristics. Conventional cancer drugscreenings depend on measurement of the amount of tumor mass, therefore,they may not necessarily select for drugs that act specifically on theCSCs. In fact, CSCs have been demonstrated to resistant to standardchemotherapies and radiotherapy, and to becoming enriched after standardanticancer treatments, which result in cancer refractory and recurrence.Methods of isolating these cells include but not limited toidentification by their ability of efflux Hoechst 33342, identificationby the surface markers these cells express, such as CD133, CD44, CD166,and others, and enrichment by their tumorigenic property. The mountingevidence linking cancer stem cells to tumorigenesis unravel enormoustherapeutic opportunity of targeting cancer stem cells.

The key to unlocking this untapped potential is the identification andvalidation of pathways that are selectively important for CSCself-renewal and survival. Though multiple pathways underlyingtumorigenesis in cancer and in embryonic stem cells or adult stem cellshave been elucidated in the past, no pathway have been identified andvalidated for CSC self-renewal and survival.

The present invention provides evidence that Stat3 pathway activity iscritical for both CSC's survival and self-renewal (Example 1). Thepresent invention further provides compounds that are effectiveinhibitors of Stat3 pathway activities (Example 2). The presentinvention also provides both in vitro and in vivo data that these Stat3inhibitors do inhibit CSCs' self-renewal and are apoptotic to CSCs(Example 3). The present invention also shows that these compounds canselectively kill a broad spectrum of cancer cells in vitro (Example 4)and inhibit a similarly broad range of cancers in vivo (Example 5).Moreover, the present invention empirically confirms Stat3 inhibitors'efficacy against metastatic cancer (Example 6). Furthermore, the presentinvention empirically confirms that these compounds can achieve adesired PK exposure for selective killing of cancer cells in vivo(Example 7).

The data provided herein, combined with recent breakthroughs in CSCresearch, allows the present invention to provide an array of methodsdirected at inhibiting CSCs, or treating cancers that have CSCs inspecific or cancers in general. Also provided herein are methodsdirected at inhibiting Stat3 pathway activity in cells, or treatingdisorders, both cancerous and non-cancerous, that are associated withaberrant Stat3 pathway activities. The present invention also providesrelated methods (e.g., manufacturing and drug candidate screening),materials, compositions and kits.

With the finding that down-regulating or blocking the Stat3 pathwayinhibits both self-renewal and survival of CSCs (Example 1), the presentinvention provides a method of inhibiting cancer stem cells where atleast some Stat3 pathway activity in the CSCs are inhibited through aStat3 pathway inhibitor. In one embodiment, most, i.e., more than 50%,of the Stat3 pathway activity is inhibited. In another embodiment,substantially all of the Stat3 pathway activity is inhibited. The methodcan prevent the CSCs from self-renewal, such that it is no longer ableto replenish its numbers by dividing into tumorigenic CSC cells. Or, themethod can induce cell death in CSCs.

This method can be used to treat a subject's cancer. Cancers that areknown to have CSCs and aberrant (e.g., overactive or constituentlyactive) Stat3 pathway activities are good candidates for such treatment,and include but are not limited to: breast cancer, head and neck cancer,lung cancer, ovarian cancer, pancreatic cancer, colorectal carcinoma,prostate cancer, renal cell carcinoma, melanoma, hepatocellularcarcinomas, cervical cancer, sarcomas, brain tumors, gastric cancers,multiple myeloma, leukemia, and lymphomas. In an embodiment, the methodis used to treat liver cancers, head and neck cancers, pancreaticcancers, and/or gastric cancers. In another embodiment, the method isused to treat multiple myeloma, brain tumors, and sarcomas.

Further, as CSCs have been demonstrated to be fundamentally responsiblefor tumorigenesis, cancer metastasis and cancer reoccurrence, anymethods of the invention directed to inhibiting CSCs can be practiced totreat cancer that is metastatic, refractory to a chemotherapy orradiotherapy, or has relapsed in the subject after an initial treatment.

In one embodiment, the inhibitor is isolated, purified or synthetic, andcan be selected from the group consisting of a small molecule Stat3inhibitor, an RNAi agent against Stat3, an antisense agent againstStat3, a peptidomimetic Stat3 inhibitor, and a G-quartetoligodeoxynucleotides Stat3 inhibitor. The inhibitor may be isolated orpurified from a natural product as well.

The mechanism of inhibition can be selected to target any step in theStat3 pathway. For example, the inhibitor can substantially inhibitphosphorylation of the Stat3 protein, substantially inhibit dimerizationof the Stat3 protein, substantially inhibit nuclear translocation of theStat3 protein, substantially inhibit DNA-binding activity of the Stat3protein, and/or substantially inhibit transcription activities of theStat3 protein. Alternatively, the Stat3 pathway inhibitor may inhibitone or more upstream or downstream components in the Stat3 pathway.

Stat3 pathway can be activated in response to cytokines, such as IL-6,or by a series of tyrosine kinases, such as EGFR, JAKs, Abl, KDR, c-Met,Src, and Her2. The downstream effectors of Stat3 include but are notlimited to Bcl-xl, c-Myc, cyclinD1, Vegf, MMP-2, and survivin (FIG. 2).Stat3 pathway is found to be aberrantly active in a wide variety ofhuman diseases, as shown in Table 1. Existing clinical samples examinedshowed that persistently active Stat3 pathway occurs in more than halfof breast and lung cancers, hepatocellular carcinomas, multiple myelomasand more than 95% of head and neck cancers. Activated Stat3 has alsobeen demonstrated in a number of autoimmune and inflammatory diseases.Furthermore, as cytokines, such as interleukin 6, mediated inflammationis the common causative origin for Atherosclerosis [38], PeripheralVascular Disease [39, 40], Coronary Artery Disease [39, 40],hypertension [41], Osteroprorosis [42], Type 2 Diabetes, and Dementia[43] and gp130-Jaks-Stats is the main pathway activated by IL-6,inhibition of Stat3 pathway may prevent these diseases as well.

TABLE 1 Activation of STAT3 PATHWAY in human diseases DISEASES REF.ONCOLOGY Solid Breast Cancer [44] DISEASES Tumors Head and Neck Cancer(SCCHN) [45] Lung Cancer [46] Ovarian Cancer [47] Pancreatic Cancer [48]Colorectal carcinoma [49] Prostate Cancer [50] Renal Cell carcinoma [51]Melanoma [52] Hepatocellular carcinomas [34] Cervical Cancer [53]Endometrial Cancer [53] Sarcomas [54, 55] Brain Tumors [56] GastricCancers [27] Hematologic Multiple Myeloma [57] Tumors LeukemiaHTLV-1-dependent Leukemia [58] Chronic Myelogenous Leukemia [51] AcuteMyelogenous Leukemia [59] Large Granular Lymphocyte [60] LeukemiaLymphomas EBV-related/Burkitt's [61] Mycosis Fungoides [51] HSVSaimiri-dependent (T-cell) [51] Cutaneous T-cell Lymphoma [62] Hodgkin'sDiseases [51] Anaplastic Large-cell Lymphoma [63] IMMUNE InflammatoryInflammatory Bowel Diseases [64] DISEASES Diseases InflammatoryArthritis [65-67] Crohn's Diseases [68] Chronic inflammatory conditions[69] Autoimmune Reumatoid Arthritis [65, 66, 7072] Systemic lupuserythematosus [73] Asthma [74] Allergy [75] Infections [76]PROLIFERATIVE Psoriasis [77] DISORDERS Keloids [78] Warts [79]Myelodysplastic syndrome [80] Polycythemia vera [81] CNS DISEASESAlzheimer's [36, 82, 83] Multiple sclerosis (MS) [36, 82, 84]

In one embodiment, the Stat3 inhibitor according to the presentinvention is: 2-(1-hydroxyethyl)-naphtho[2,3-b]furan-4,9-dione,2-acetyl-7-chloro-naphtho[2,3-b]furan-4,9-dione,2-acetyl-7-fluoro-naphtho[2,3-b]furan-4,9-dione,2-acetylnaphtho[2,3-b]furan-4,9-dione,2-ethyl-naphtho[2,3-b]furan-4,9-dione, phosphoric acidmono-[1-(4,9-dioxo-3a,4,9,9a-tetrahydro-naphtho[2,3-b]furan-2-yl)-vinyl]ester,phosphoric acid1-(4,9-dioxo-3a,4,9,9a-tetrahydro-naphtho[2,3-b]furan-2-yl)-vinyl esterdimethyl ester, an enantiomer, diastereomer, tautomer, and a salt orsolvate thereof (the “Compound of the Invention”) (Example 2). Thepresent invention also provides both in vitro and in vivo data that theCompound of the Invention inhibits CSCs' self-renewal and inducesapoptosis in CSCs (Example 3).

Having provided evidence that downregulation of Stat3 pathway inhibitsCSCs, the present invention provides a method of identifying a drugcandidate capable of inhibiting a cancer stem cell. The method comprisesscreening for a drug candidate that inhibits Stat3 pathway activity. Invarious embodiments, the drug candidate is a small molecule Stat3inhibitor, an RNAi agent against Stat3, an antisense agent againstStat3, a peptidomimetic Stat3 inhibitor, or a G-quartetoligodeoxynucleotide Stat3 inhibitor.

In one embodiment, the drug candidate is capable of inducing cell deathin CSC or at least inhibiting its self-renewal. Various phases in thepathway can be targeted for screening the drug candidate. For example,various embodiments of the method can screen for drug candidates thatsubstantially inhibits phosphorylation of the Stat3 protein,substantially inhibits dimerization of the Stat3 protein, substantiallyinhibits nuclear translocation of the Stat3 protein, substantiallyinhibits DNA-binding activities of the Stat3 protein, or substantiallyinhibits transcription activities of the Stat3 protein.

As Example 2 below shows, the Compound of the Invention inhibits Stat3transcription activity, and Stat3 DNA-binding activity in vitro. Example2 further shows that the Compound of the Invention inhibits in vivo boththe expression of Stat3 downstream effectors (e.g., cyclin D1 andsurvivin) and Stat3 DNA binding activity.

Accordingly, in another aspect, the present invention provides a methodof inhibiting cellular Stat3 pathway activity where an effective amountof the Compound of the Invention is administered. In another aspect, theCompound of the Invention can be used to formulate a pharmaceuticalcomposition to treat or prevent disorders or conditions associated withaberrant Stat3 pathway activities. A disorder is considered herein“associated with” aberrant Stat3 pathway activities if a patientsuffering from the disorder, which can be a subtype within a type ofdisorder, typically have in at least some of the patient's cellsaberrant Stat3 pathway activity, which can, but not necessarily,contribute to the pathology of the disorder. Some of the disorders knownto be associated with aberrant Stat3 pathway activities include but arenot limited to: autoimmune diseases, inflammatory diseases, inflammatorybowel diseases, arthritis, autoimmune demyelination disorder,Alzheimer's disease, stroke, ischemia reperfusion injury and multiplesclerosis. Some of the disorders known to be associated with aberrantStat3 pathway activities are cancers and include but are not limited to:various types of breast cancers, head and neck cancers, lung cancers,ovarian cancers, pancreatic cancers, colorectal carcinoma, prostatecancers, renal cell carcinoma, melanoma, hepatocellular carcinomas,cervical cancers, sarcomas, brain tumors, gastric cancers, multiplemyeloma, leukemia, and lymphomas.

Related to this aspect of the invention, a kit is provided that includesone or more agents for diagnosing a disorder associated with aberrantStat3 pathway activities, and a therapeutically effective amount of theCompound of the Invention or another effective Stat3 pathway inhibitor.The diagnostic agent can be any suitable reagent depending on thesuspected disorder, and may include agents needed to draw a bloodsample, take a biopsy, screen for a biomolecule (e.g., antigen orantibody) or extract genetic information from a sample. The agent mayinclude a solvent, a detergent, an anticoagulant, an antigen, anantibody, an enzyme, a PCR primer, and so on.

Whether or not a patient is diagnosed with a disorder known to implicateaberrant Stat3 activity, a physician can always order a test to see ifthere is aberrant Stat3 activity in a biopsy sample taken from thepatient. Therefore, a kit is provided that includes: one or more agentsfor diagnosing aberrant Stat3 pathway activities, and a therapeuticallyeffective amount of the Compound of the Invention or another effectiveStat3 pathway inhibitor. In one feature, aberrant Stat3 pathway activitycan be identified through any suitable analytical means to examine anyindicia of such activity, e.g., expression (level, duration, etc.) ofphosphorylated Stat3 or of a surrogate upstream or downstream regulatorof Stat3 phosphorylation. Similar to the kit described previously, thediagnostic agent can be any suitable reagent depending on the indicia ofaberrant Stat3 pathway activity that the test looks at.

As Example 3 below shows, the Compound of the Invention, which is atleast a Stat3 pathway inhibitor, kills cancer stem cells. Example 3 alsodemonstrates that the Compound of the Invention also inhibits CSCspherogenesis, an indication of successful inhibition of CSCself-renewal, both in vitro and in vivo.

Accordingly, in an aspect, the present invention provides a method ofinhibiting cancer stem cells where an effective amount of the Compoundof the Invention is administered to the cells. Cancers known to haveCSCs are good candidates for such treatments, and include but are notlimited to: various types of breast cancers, head and neck cancers, lungcancers, ovarian cancers, pancreatic cancers, colorectal carcinoma,prostate cancers, liver cancers, melanoma, multiple myeloma, braintumors, sarcomas, medulloblastoma, and leukemia.

Further, as CSCs have been demonstrated to be fundamentally responsiblefor tumorigenesis, cancer metastasis and cancer reoccurrence, anymethods of the invention directed to inhibiting CSCs can be practiced totreat cancer that is metastatic, refractory to a chemotherapy orradiotherapy, or has relapsed in the subject after an initial treatment.Example 6 below specifically tests in vivo the anti-metastasis efficacyof the Compound of the Invention, and the data show significantreduction in the number of primary tumor foci and the spontaneous livermetastasis.

In Example 4 below, the Compound of the Invention is not only shown tocause apoptosis in a broad spectrum of cancer cells, but also to exhibitselectivity in its cytotoxicity which is critical for developinglow-toxicity therapeutics. Selective cytotoxicity as used herein refersto a compound's ability to kill cancer cells while substantially sparingnormal cells, sometimes under certain conditions. Normal cells usuallyrefer to healthy, non-tumorigenic cells. Conditions that result inselective cytotoxicity for a drug candidate are hard to predict becausethey require knowledge of the underlying mechanism of cytotoxicity. Forexample, to lower the toxicity of an anti-cancer drug that targetsmicrotubule formation during mitosis presents quite different factors towork with than a drug that blocks cellular metabolic processes. Asuitable condition for engendering selective cytotoxicity needs tobalance the need for the drug to be toxic enough to effectively killcancer cells while tolerable enough to normal cells. For instance, iflower concentration is used, that often means prolonged infusion isneeded to kill cancer cells.

From data generated in the examples of this invention including thoseshown in Example 4, it appears that selective cytotoxicity can beachieved for the Compound of the Invention if affected cells are notexposed to a critical concentration of the compound continuously beyonda certain duration. In a method aimed at selectively killing cancercells in a subject, a pharmaceutical composition that has the Compoundof the Invention is administered to the subject such that the compoundconcentration in the subject's plasma is not maintained above a criticalconcentration for more than 24 hours after each dose. This method can beused to treat all cancers, including any of the groups of cancersdescribed here, and to treat Stat3-associated disorder, an exemplarylist of which is already provided above and is not repeated here.Alternatively, the duration can be further restricted to 12, 16, and 20hours after each dose. The critical concentration for each compound mayvary. In various embodiments of the present invention, the criticalconcentration is about 100 μM, about 50 μM, about 30 μM, or about 20 μM.

In one embodiment of the method, the cancer being treated is selectedfrom the following group: liver cancer, head and neck cancer, pancreaticcancer, gastric cancer, renal cancer, sarcoma, multiple myeloma,metastatic breast cancer, metastatic prostate cancer, leukemia,lymphoma, esophageal cancer, brain tumor, glioma, bladder cancer,endometrial cancer, thyroid cancer, bile duct cancer, bone cancer, eyecancer (retinoblastoma), gallbladder cancer, pituitary cancer, rectalcancer, salivary gland cancer, and nasal pharyngeal cancer.

In an aspect, the present invention provides a method of treating cancerin a subject, where a therapeutically effective amount of apharmaceutical composition comprising the Compound of the Invention isadministered to the subject. The cancer may be metastatic. The subjectmay be a mammal, e.g., a human being.

For any of the methods of treating a subject described herein, thepresent invention provides effective dosing ranges, dosing frequencies,and plasma concentrations of the compounds. In various embodiments, thepharmaceutical composition is administered at a dosage: (a) from about 1mg/m² to about 5,000 mg/m² (I.V.) or from about 1 mg/m² to about 50,000mg/m² (PO); (b) from about 2 mg/m² to about 3,000 mg/m² (I.V.) or fromabout 10 mg/m² to about 50,000 mg/m² (PO). In various embodiments, thecompound of the present invention can be administered every other day(Q2D), daily (QD), or twice a day (BID). In one embodiment, thepharmaceutical composition is administered orally and no more than fourtimes a day (QID).

In one feature, the pharmaceutical composition is administered to thesubject such that the compound concentration in the subject's plasma isnot maintained above a critical concentration for more than 24 hours (or12, 16, and 20 hours) after each dose. According to alternativeembodiments of the invention, the plasma concentration of the compounddoes not exceed the critical concentration at a certain time point aftereach does, e.g., 12, 16, 20, or 24 hours, as a regimen that avoidsnon-selective toxicity. In various embodiments of the present invention,the critical concentration is about 100 μM, about 50 μM, about 30 μM, orabout 20 μM. The compositions, in certain cases, are isolated, purifiedor synthesized.

In another aspect, the present invention provides a pharmaceuticalcomposition that comprises the Compound of the Invention, and apharmaceutically-acceptable excipient, carrier, or diluent. In onefeature, the composition is suitable for oral, nasal, topical, rectal,vaginal or parenteral administration, or intravenous, subcutaneous orintramuscular injection.

Formulations of the present invention include those suitable for oral,nasal, topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which canbe combined with a carrier material to produce a single dosage form willvary depending upon the mammal being treated and the particular mode ofadministration. The amount of active ingredient, which can be combinedwith a carrier material to produce a single dosage form, will generallybe that amount of the compound which produces a therapeutic effect.Generally, out of 100%, this amount will range, for example, from about1% to about 99% of active ingredient, from about 5% to about 70%, fromabout 10% to about 30%.

Therapeutic compositions or formulations of the invention suitable fororal administration may be in the form of capsules, cachets, pills,tablets, lozenges (using a flavored basis, usually sucrose and acacia ortragacanth), powders, granules, or as a solution or a suspension in anaqueous or non-aqueous liquid, or as an oil-in-water or water-in-oilliquid emulsion, or as an elixir or syrup, or as pastilles (using aninert base, such as gelatin and glycerin, or sucrose and acacia) and/oras mouth washes and the like, each containing a predetermined amount ofthe Compound of the Invention as an active ingredient. The Compound ofthe Invention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theCompound of the Invention is mixed with one or morepharmaceutically-acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: fillers or extenders,such as starches, lactose, sucrose, glucose, mannitol, and/or silicicacid; binders, such as, for example, carboxymethylcellulose, alginates,gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; humectants, suchas glycerol; disintegrating agents, such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,sodium carbonate, and sodium starch glycolate; solution retardingagents, such as paraffin; absorption accelerators, such as quaternaryammonium compounds; wetting agents, such as, for example, cetyl alcohol,glycerol monostearate, and polyethylene oxide-polypropylene oxidecopolymer; absorbents, such as kaolin and bentonite clay; lubricants,such a talc, calcium stearate, magnesium stearate, solid polyethyleneglycols, sodium lauryl sulfate, and mixtures thereof; and coloringagents. In the case of capsules, tablets and pills, the pharmaceuticalcompositions may also comprise buffering agents. Solid compositions of asimilar type may also be employed as fillers in soft and hard-filledgelatin capsules using such excipients as lactose or milk sugars, aswell as high molecular weight polyethylene glycols and the like.

Liquid dosage forms for oral administration of the Compound of theInvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, com, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof. Additionally, cyclodextrins,e.g., hydroxypropyl-.beta.-cyclodextrin, may be used to solubilizecompounds.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents. Suspensions, inaddition to one or more Compounds of the Invention, may containsuspending agents as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more Compounds of the Invention,with one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active pharmaceutical agents of theinvention. Formulations of the present invention which are suitable forvaginal administration also include pessaries, tampons, creams, gels,pastes, foams or spray formulations containing such carriers as areknown in the art to be appropriate.

Dosage forms for the topical or transdermal administration of acomposition according to the invention include powders, sprays,ointments, pastes, creams, lotions, gels, solutions, patches andinhalants. The active compound may be mixed under sterile conditionswith a pharmaceutically-acceptable carrier, and with any preservatives,buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to theCompound of the Invention, excipients, such as animal and vegetablefats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds according to the inventionin combination with one or more pharmaceutically-acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

In some cases, in order to prolong the effect of the compositionaccording to the invention, it is desirable to slow its absorption bythe body from subcutaneous or intramuscular injection. This may beaccomplished by the use of a liquid suspension of crystalline oramorphous material having poor water solubility. The rate of absorptionof the drug then depends upon its rate of dissolution, which, in turn,may depend upon crystal size and crystalline form. Alternatively,delayed absorption of a parenterally-administered composition isaccomplished by dissolving or suspending the compound in an oil vehicle.One strategy for depot injections includes the use of polyethyleneoxide-polypropylene oxide copolymers wherein the vehicle is fluid atroom temperature and solidifies at body temperature.

The pharmaceutical compounds of this invention may be administered aloneor in combination with other pharmaceutical agents, or with otheranti-cancer therapies as described herein, as well as in combinationwith a pharmaceutically-acceptable excipient, carrier, or diluent.

In an embodiment, the pharmaceutically acceptable excipient, carrier, ordiluent comprises a lipid for intravenous delivery. The lipid can be:phospholipids, synthetic phophatidylcholines, naturalphophatidylcholines, sphingomyelin, ceramides, phophatidylethanolamines,phosphatidylglycerols, phosphatidic acids, cholesterol, cholesterolsulfate, and hapten and PEG conjugated lipids. The lipid may be in theform of nanoemulsion, micelles, emulsions, suspension, nanosuspension,niosomes, or liposomes. In an embodiment, the pharmaceuticallyacceptable excipient, carrier, or diluent is in a form of micellaremulsion, suspension, or nanoparticle suspension, and it furthercomprises an intravenously acceptable protein, e.g., human albumin or aderivative thereof, for intravenous delivery.

In an embodiment, the pharmaceutically acceptable excipient, carrier, ordiluent comprises a waxy material for oral delivery. The waxy materialmay be mono-, di-, or tri-glycerides, mono-, di-fatty acid esters ofPEG, PEG conjugated vitamin E (vitamin E TPGs), and/or Gelucire. TheGelucire can be selected from Gelucire 44/14, Gelucire 43/01, Gelucire50/02, Gelucire 50/13, Gelucire 37/02, Gelucire 33/01, Gelucire 46/07,and Gelucire 35/10. In an embodiment, the pharmaceutically acceptableexcipient, carrier or diluent is selected from capryol, transcutol hp,labrafil M, labrasol, triacetin, pharmasolv, ethanol, poly vinylpyrrolidine, carboxymethyl cellulose, tween 20, and tween 80. In anembodiment, the pharmaceutically acceptable excipient, e.g., Gelucire44/14, is mixed with a surfactant, which can be Tween 80 or Tween 20.These embodiments of pharmaceutical compositions can be furtherformulated for oral administration.

The Compound of the Invention can be synthesized using commerciallyavailable starting materials and processes well known to one skilled inthe art of organic chemistry. In Examples 8-10, the present inventionprovides a manufacturing process for some of the claimed compounds.

According to one or more embodiments of the present invention, a smallmolecule Stat3 inhibitor refers to any low molecular-weight drug thatshows inhibitory activity against Stat3. Compared to larger molecularweight pharmaceuticals such as proteins, peptides, and carbohydrates,small molecules can more easily penetrate cell membranes and the bloodbrain barrier. These molecules tend to incur lower process developmentand manufacturing costs.

According to one or more embodiments of the present invention, an RNAitherapy is the direct use of RNA interference (RNAi) in the treatment ofdiseases by silencing genes that give rise to bad proteins and,therefore, disease. RNAi is a naturally occurring process thatsuppresses certain gene activity in living cells. It is a widelyconserved eukaryotic function that double stranded RNA triggers in cellsvia short RNA duplex intermediates such as small interfering RNAs(siRNAs). Through a series of processing steps, one of the two strandsof the siRNA complexes with proteins to form RISC (RNA-induced silencingcomplex). RISC recognizes the complementary RNA sequence by Watson-Crickbase pairing and then cleaves it. RNAi also include shRNA, miRNA, andothers.

According to one or more embodiments of the present invention, anantisense therapy is a form of treatment for genetic disorders orinfections. When the genetic sequence of a particular gene is known tobe causative of a particular disease, it is possible to synthesize astrand of nucleic acid (DNA, RNA or a chemical analogue) that will bindto the messenger RNA (mRNA) produced by that gene and inactivate it,effectively turning that gene “off” This is because mRNA has to besingle stranded for it to be translated. This synthesized nucleic acidis termed an “anti-sense” oligonucleotide because its base sequence iscomplementary to the gene's messenger RNA (mRNA), which is called the“sense” sequence (so that a sense segment of mRNA “5′-AAGGUC-3′” wouldbe blocked by the anti-sense mRNA segment “3′-UUCCAG-5′”).

According to one or more embodiments of the present invention, apeptidomimetic is a small protein-like chain designed to mimic apeptide. They typically arise from modification of an existing peptidein order to alter the molecule's properties. For example, they may arisefrom modifications to change the molecule's stability or biologicalactivity. This can have a role in the development of drug-like compoundsfrom existing peptides. These modifications involve changes to thepeptide that will not occur naturally (such as altered backbones and theincorporation of non-natural amino acids).

According to one or more embodiments of the present invention, G-quartetoligodeoxynucleotide inhibitors are catalytic DNA molecules (DNAzymes)designed to inhibit proteins independent of their RNA-cleavage activityin cells.

Materials and Methods Biological Assays

Compounds of the present invention can be tested according to theprotocol described above. Table 2 shows the list of compounds describedin the protocol.

TABLE 2 Compound Name Compound Code2-(1-hydroxyethyl)-naphtho[2,3-b]furan-4,9-dione 3012-Acetyl-7-Chloro-naphtho[2,3-b]furan-4,9-dione 4162-Acetyl-7-Fluoro-naphtho[2,3-b]furan-4,9-dione 4182-acetylnaphtho[2,3-b]furan-4,9-dione 4012-ethyl-naphtho[2,3-b]furan-4,9-dione 101 phosphoric acidmono-[1-(4,9-dioxo- 4011 3a,4,9,9a-tetrahydro-naphtho[2,3-b]furan-2-yl)-vinyl] ester phosphoric acid 1-(4,9-dioxo-4012 3a,4,9,9a-tetrahydro-naphtho[2,3- b]furan-2-yl)-vinyl esterdimethyl ester

Cell Culture:

HeLa, DU145, H1299, DLD1, SW480, A549, MCF7, LN18, HCT116, HepG2, Paca2,Panc1, LNcap, FaDu, HT29, and PC3 cells (ATCC, Manassas, Va.) weremaintained in Dulbecco's Modified Eagle Medium (DMEM) (Invitrogen,Carlsbad, Calif.) supplemented with 10% fetal bovine serum (FBS) (GeminiBio-Products, West Sacramento, Calif.) and 5%penicillin/streptomycin/amphotercin B (Invitrogen).

Hoechst Side Population:

To identify and isolate side population (SP) and non-SP fractions, SW480cells were removed from the culture dish with trypsin and EDTA, pelletedby centrifugation, washed with phosphate-buffered saline (PBS), andresuspended at 37° C. in Dulbecco's modified Eagle's medium (DMEM)containing 2% FBS and 1 mM HEPES. The cells were then labeled withHoechst 33342 (Invitrogen) at a concentration of 5 μg/mL. The labeledcells were incubated for 120 minutes at 37° C., either alone or with 50μM verapamil (Sigma-Aldrich, St. Louis). After staining, the cells weresuspended in Hanks' balanced saline solution (HBSS; Invitrogen)containing 2% FBS and 1 mM HEPES, passed a through 40 μm mesh filter,and maintained at 4° C. until flow cytometry analysis. The Hoechst dyewas excited at 350 nm, and its fluorescence was measured at twowavelengths using a 450 DF10 (450/20 nm band-pass filter) and a 675LP(675 nm long-pass edge filter) optical filter. The gating on forward andside scatter was not stringent, and only debris was excluded [15],

CSC Isolation with Surface Markers:

Sorting tumor cells based primarily upon the differential expression ofthe surface marker(s), such as CD44 or CD133, have accounted for themajority of the highly tumorigenic CSCs described to date. CD133isolation is based upon the method of Ricci-Vitiani et al. [20], withslight modification. CD133⁺ cells were isolated by either fluorescenceactivated cell sorting (FACS) or magnetic nanoparticle-based separation.Briefly, 10′ cells/mL were labeled with CD133/1 (AC133)-PE forFACS-based cell sorting; or with CD133/1 (AC133)-biotin (MiltenyiBiotec, Auburn, Calif.) for magnetic field-based separation using theEasySep® biotin selection kit (Miltenyi Biotec) according to themanufacturer's recommendations. Non-specific labeling was blocked withthe supplied FcR blocking reagent and antibody incubations (1:11) werecarried out on ice for 15 minutes in PBS with 2% FBS and 1 mM EDTA. Fivewashes were done for EasySep® isolation, whereas cells were pelleted at400×g for 5 minutes and resuspended at 2×10⁷/mL, before sorting by FACS.

CD44^(high) cells were isolated by FACS according to the methodsdescribed in Ponti et al, with slight modification [85]. Briefly, aftertrypsinization and recovery of cells for 30 minutes at 37° C. in growthmedia, cells were pelleted at 400×g and were resuspended in PBS with 2%FBS and 1 mM EDTA at 1×10⁶ cells/mL. Cells were then incubated on icewith a 1:100 dilution of CD44-FITC (BD Biosicences, San Diego, Calif.)for 15 minutes. Alternatively, CD24-PE (BD Bioscences, San Diego,Calif.) (1:100) was utilized for negative selection. After washing threetimes, cells were resuspended at 2×10⁶/mL and passed through a 40 μMmesh before sorting.

Sphere Assay:

A reliable method of measuring the self-renewal capacity of cellpopulation if the ability to be cultured as spheres in the absence ofserum or attachment. CD44^(high) FaDu or Hoechst side population cancerstem cells were cultured in ultra low attachment plates in cancer stemcell media (DMEM/F12, B27 Neurobasal supplement, 20 ng/ml EGF, 10 ng/mlFGF, 4 μg/ml insulin, and 0.4% BSA) to allow spheres formation.Typically, sphere formation was evaluated by microscopy after 10-14 daysin culture and spheres with >50 cells were scored.

Luciferase Reporter Assay:

HeLa Cells were co-transfected with Stat3-luciferase (Stat3-Luc)reporter vector (Panomics, Fremont, Calif.) and Renilla luciferase(Promega, Madison, Wis.) using Lipofectamine 2000 as described by themanufacturer (Invitrogen). Following transfection, cells were maintainedin medium containing 0.5% FBS for 24 hours. Cells were then treated withthe indicated compound for 30 minutes prior to the addition of 25 ng/mloncostatin M (OSM) (R&D Systems, Minneapolis, Minn.) to the medium. 6hours following OSM addition, cells were harvested and levels of fireflyand renilla luciferase were measured using the Dual-Glo Luciferase AssaySystem as described by the manufacturer (Promega).

Analysis of Apoptosis:

Cells treated with or without compound were harvested at 5 hours posttreatment for Annexin-V staining. Collected cells were washed with PBSand resuspended in Annexin-V-FITC containing buffer and stainedaccording to manufactures directions (Roche). Annexin-V positive cellswere determined by Flow cytometry.

STAT3 DNA Binding Assay:

Electrophoretic mobility shift assay (EMSA) was performed as describedby the manufacturer (Li-Cor Biosciences, Lincoln, Nebr.). Briefly,nuclear extracts were made from HeLa cells using the NucBuster ProteinExtraction Kit as described by the manufacturer (EMD Biosciences, SanDiego, Calif.). 5 μg of nuclear extract was pre-incubated with theindicated dose of indicated compound for 30 minutes prior to a 15-minuteincubation with the IR700-labeled consensus Stat3 oligonucleotide.Samples were then electrophoresed on a polyacrylamide gel and directlyscanned using the Odyssey infrared imaging system (Li-Cor Biosciences).For the enzyme-linked immunosorbent assay (ELISA), 5 μg of nuclearextract was preincubated with indicated concentration of indicatedcompound for 30 minutes prior to the addition of biotinylated oligo(5′-Biotin-GATCCTTCTGGGAATTCCTAGATC-3′, SEQ ID NO. 1). Stat3-DNAcomplexes were then captured on streptavidin coated 96 well plates(Pierce, Rockford, Ill.). Bound complexes were then incubated with Stat3polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.)followed by anti-rabbit HRP conjugated secondary antibody (GEHealthcare, Pittsburgh, Pa.). Bound antibody was then visualized byaddition of TMB substrate (Pierce) and absorbance measured at 450 nm,

Cell Viability Determination:

For 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT)(Sigma-Aldrich, St. Louis, Mo.) analysis, cells were plated in 96 wellplates at 10,000 cells per well. 24 hours after plating, compound wasadded to cells at indicated doses. 22 hours following compound addition,MTT was added to each well (0.5 mg/ml, final concentration) and plateswere incubated for an additional 2 hours at 37° C. Medium was thenaspirated and the formazan product was solubilized in 100 μl ofisopropyl alcohol. The absorbance of each well was measured at 570 nmusing a microplate reader.

Immunofluorescence:

Cells treated with indicated compound for an indicated time were eitherfixed in 4% formaldehyde or cold methanol for the detection of AnnexinV, cleaved caspase 3, or stat3, respectively. Coverslips were air driedand rehydrated in PBS at room temperature for 10 min. Samples were thenincubated in blocking buffer (PBS, 5% FBS) for 10 min at roomtemperature in a humid chamber. Cells were incubated overnight at 4° C.with primary antibodies. After washing, the cells were incubated for 1hour at room temperature with a 1:500 dilution of FITC conjugatedanti-rabbit antibody. Images were captured with a Nikon TE200 microscopeequipped with epifluorescence and a SPOT mosaic CCD camerapolyclonalAnti-cleaved caspase 3 antibody (1:100) was obtained from Cell SignalingTechnology, Danvers, Mass. Annexin-V-FITC was obtained from Roche,Penzberg, Germany. Polyclonal anti-Stat3 antibody was obtained fromSanta Cruz.

Gene knockdown by TPIV® technology:

The TPIV® (Therapeutic Pathway Identification and Validation) technology(Boston Biomedical Inc., Norwood, Mass., USA) provides plasmids that canbe used to first transfect bacteria that are in turn taken up by amammalian subject. After bacterial lysis, dsRNA encoded by the TPIV®plasmids and processed by the bacteria get released into the mammaliancell cytoplasm and effect targeted gene knockdown. The TPIV® technologyis described in co-owned PCT patent application no. PCT/US08/68866 filedon Jun. 30, 2008, the entire content of which is incorporated herein byreference. Specifically, a TPIV® plasmid that encodes effective siRNAsequences against Stat3 was constructed by PCR-cloning of a Stat3plasmid purchased from Origene Technologies (Rockville, Md., USA) usingthe following primers:

TPIV-Stat3 (300 bp insert) Primers: Stat3 TPIV For (SEQ ID NO. 2)5′-GGATCTAGAATCAGCTACAGCAGC Stat3 TPIV Rev (SEQ ID NO. 3)5′-TCCTCTAGAGGGCAATCTCCATTG

The control plasmid is constructed using a pGL2 plasmid purchased fromPromega (Madison, Wis., USA).

TPIV-GL2 (300 bp insert) Primers: GL2 TPIV For (SEQ ID NO. 4)5′-CCCTCTAGATGGTTCCTGGAAC GL2 TPIV Rev (SEQ ID NO. 5)5′-GCTCTAGAAACCCCTTTTTGG

Chemically competent E. coli BL21 (DE3) pLYSe bacteria (50˜100 μl) weretransformed with control or 100 ng of Stat3-targeting TPIV® plasmidaccording to the manufacturer instructions (Stratagene). A single colonywas then inoculated into BHI medium containing 100 μg/ml ampicillin, andgrown overnight at 37° C. The next day, 5 ml of each overnight culturewas diluted 1:40 into fresh BHI medium containing 100 μg/ml ampicillinand grown for a further 2-4 hours (until the OD₆₀₀=0.5). Each culturewas then treated with IPTG (1 mM final concentration) for 2-4 hours toinduce transcription of the long double strand RNAs which would beprocessed into a cocktail siRNAs by the bacteria. After IPTG induction,the total number of bacteria in each culture was calculated by measuringthe OD₆₀₀ value (8×10⁸ bacteria/ml culture has an OD₆₀₀=1). The numberof bacteria for cell treatment was then calculated according to cellconfluency and the needed multiplicity of infection (MOI; try ranges of20:1 to 2000:1, bacteria to cells) in an appropriate reaction volume. Asa rule of thumb, the reaction volume should be chosen to result in3×10⁸/ml for a 1000:1 MOI. The required volume of bacteria culture wasthen centrifuged at 2500 g for 10 mins at 4° C. and the pellet waswashed once with serum-free culture medium that was used for the cellsbeing bactofectioned plus 100 μg/ml ampicillin and 1 mM of IPTG, andresuspended in the same medium at the required density for bacterialinfection (bactofection).

At the same time, cancer cells or cancer stem cells were isolated. 30minutes before bactofection, the cell culture medium was replaced with 2ml of fresh serum-free medium containing 100 μg/ml of ampicillin and 1mM IPTG. Bacteria prepared above were then added to the cells at thedesired MOI for 2 hours at 37° C.

After the infection period, the cells were washed 3 times usingserum-free cell culture medium. The cells were then incubated with 2 mlof fresh complete cell culture medium containing 100 μg/ml of ampicillinand 150 μg/ml of gentamycin for 2 hours to kill any remainingextracellular bacteria. After treatment with ampicillin and gentamycin,the cells were incubated with 3 ml of fresh complete RPMI 1640 mediumcontaining 10 μg/ml of ofloxacin to kill any intracellular bacteria. Thecells were then harvested or analysis at various time points in order toassess the extent of target gene silencing and the resulting phenotypes.

In Life Evaluations:

Daily examinations into the health status of each animal were alsoconducted. Body weights were checked every three days. Food and waterwas supplied daily according to the animal husbandry procedures of thefacility. Treatment producing >20% lethality and or >20% net body weightloss were considered toxic. Results are expressed as mean tumor volume(mm³) ±SE. P Values <0.05 are considered to be statistically relevant.

Animal Husbandry:

Male or female athymic nude mice 4-5 weeks (Charles River Laboratories,Wilmington, Mass.), were acclimated to the animal housing facility forat least 1 week before study initiation. All of the experimentalprocedures utilized were consistent with the guidelines outlined by theAmerican Physiology Society and the Guide for the Care and Use ofLaboratory Animals and were also approved by the Institutional AnimalCare and Use Committee of Boston Biomedical Inc. The animals were housedin groups of four in wood chip bedded cages in a room having controlledtemperature (68° F.-72° F.), light (12-h light-dark cycle), and humidity(45-55%). The animals were allowed free access to water and food duringthe experiment.

Intrasplenic-Nude Mouse Model System (ISMS Model):

The female nude mice were anesthetized and under aseptic conditions, anincision was made in the left flank to expose the spleen. One millionhuman colon cancer HT29 cells in 0.1 ml PBS were injected under thespleen capsule using a 27-gauge needle. The spleen was replaced in theperitoneal cavity and the incision was closed. Treatment started thenext day after the implantation till the examination day. The regimen ofthe treatments is 5 qd/wk via i.p. The mice were sacrificed whenmoribund or 30 days after the injection. The spleen and liver wereremoved and examined, and the number of tumor lesions was recorded.

Example 1 Identification of Stat3 as an Anti-Cancer Stem Cell Target

Stat3 knockdown in CSCs induces apoptosis. To determine whether cancerstem cells expressed Stat3 and whether Stat3 was constitutively active,we performed immunofluorence microscopy, which allows not only theanalysis of rare cell populations, but also provides additionalinformation on protein localization and the ability to correlatestaining with phenotype (i.e. apoptosis). Following immunofluorescentdetection of p-Stat3 and Stat3 in NSP and SP cells isolated by FACS fromSW480 colon cancer cells, we determined that Stat3 was indeed present inSP cells and that it was modestly enriched in the nucleus (FIG. 3A). Inaddition, we also observed increased p-Stat3 staining in SP cells overNSP cells, suggesting that SP cells may rely more heavily on Stat3 forsurvival.

The status of Stat3 was also evaluated in CD133⁺ cells isolated fromFaDu human head and neck cancer cells and LN18 human glioblastoma cells.As shown in FIG. 3B, Stat3 are also constitutively active in thesecells. Taken together, these data suggest Stat3 as a target that isparticularly important for cancer stem cells.

We next tested the effect of Stat3 knockdown in CSCs using TPIV®.Immunofluorescence analysis revealed that significant depletion of Stat3could be achieved within 24 hours of infection (FIG. 4A) on freshlyisolated CSCs (SP) and found that the majority of cells treated withStat3-targeting TPIV® plasmids underwent apoptosis within 24 hours ofinfection, whereas control TPIV® plasmids did not induce apoptosis tolevels above control, uninfected cells (FIG. 4B). These data demonstratethat cancer stem cells depend upon Stat3 for survival.

Knock Down of Stat3 in CSCs Inhibits CSC Spherogenesis.

CD44^(high)/CD24^(low) FaDu or Hoeschst side population cancer stemcells were isolated by FACS, and cultured in ultra low attachment platesin cancer stem cell media (DMEM/F12, B27 Neurobasal supplement, 20 ng/mLEGF, 10 ng/mL FGF, 4 μg/mL insulin, and 0.4% BSA) to allow sphereformation. Primary spheres were collected, disaggregated with trypsin,and distributed to 96-well ultra low attachment plated prior to TPIV®treatment. Bacteria were administered at an MOI of 1000 for two hoursbefore addition of anti-biotic cocktail (penstrep, gentamycin,oflaxacin). Sphere formation was assessed after 10-14 days in culture.Representative sphere images were captured before (FIG. 5, left upperpanels) or after the addition of trypan blue to identify dead cells(FIG. 5, left bottom panel). Relative spherogenesis was shown in theright panel of FIG. 5. The data clearly showed that Stat3 knockdown incancer stem cells inhibits sphereogenesis, demonstrating that Stat3 is akey self-renewal factor of cancer stem cells.

Example 2

Identification of Compounds that Inhibit Stat3 Pathway Activity

Inhibition of Stat3 Transcription Activity.

Compounds were tested for their ability to inhibit Stat3 transcriptionactivation activity in cells using a Stat3-luciferase (Stat3-luc)reporter construct. Cells transfected with Stat3-luc were cultured inreduced serum medium prior to addition of indicated compound for 30minutes. Cells were then stimulated with 25 ng/ml oncostatin M (OSM) for6 hours followed by detection of Stat3-luc reporter activity. Incubationof cells with compound 401 inhibited OSM-stimulated Stat3 reporteractivity (FIG. 6, left panel). AG490, a known inhibitor of the Jak-Statpathway, was included as a positive control for Stat3 inhibition.Etoposide, included as a control for genotoxic activity, showed littleor no Stat3 inhibition. Compound 1001, which is naphthalene instead ofnaphthoquinone as the compounds in this invention, did not inhibitOSM-stimulated Stat3 reporter activity even at a much higherconcentration (FIG. 6, right panel).

Additional compounds were tested in the Stat3 luciferase reporter assaysand the results are summarized in Table 3.

TABLE 3 Compound # IC₅₀ in Stat3-Luc assays 401 ~0.25 μM 416 ~0.75 μM418 ~0.75 μM 301 ~2 μM

Inhibition of Stat3 DNA-Binding Activity.

Nuclear extracts from HeLa cells, which contain constitutively activatedStat3 as detected by phoshporylation at the tyrosine 705 residue, wereused to perform Stat3 EMSAs to monitor Stat3 DNA binding activity.Nuclear extracts were incubated with indicated compound prior toincubation with IR700-labeled Stat3 consensus oligonucleotide. Bindingof Stat3 to the oligonucleotide was monitored by gel electrophoresis anddetection using a LiCor Odyssey infrared scanner. The Stat3 retardedband was identified and confirmed by supershift with the anti-Stat3antibody (FIG. 7A, left panel) and dose-dependent inhibition with theStat3 peptide (FIG. 7A, middle panel). Dose dependent inhibition ofStat3 DNA binding was observed following incubation of the labeled probewith compound 401 (FIG. 7A, right panel).

Additional compounds were tested in the EMSA assays. As shown in FIG.7B, compounds 401, 416 and 418 can inhibit Stat3's DNA binding activity.

Example 3

Identification of Compounds that Target Cancer Stem Cells

Identification of Compounds that are Apoptotic to Cancer Stem Cells.

Since cancer stem cells have been demonstrated to actively expelHoechst, SW480 cells were stained with Hoechst and the side population(shown in FIG. 8A, left panel gated area) was sorted out to enrich thecancer stem cells. To confirm that this side population is enriched withcancer stem cells, a control set of SW480 cells were first treated withVerapamil, an inhibitor of ABC transporters, before stained withHoechst. As shown in the right panel of FIG. 8A, Verapamil treatmentresults in the loss of the side population.

The IC₅₀ of compound 401 against the Hoechst side population wasaccessed in MTT assays and was compared to the IC₅₀ against the non-sidepopulation. The results show that the side population is as sensitive asthe non-side population to compound 401 (FIG. 8B, right panels).However, the side population is much more resistant than the non-sidepopulation to Doxorubicin (FIG. 8B, left panels), which is consistentwith previous publications [7, 86]. These data suggest that compound 401kills cancer stem cells.

The Hoechst side population cells were treated with compound 401 and themode of cell death was accessed by Annexin V (an early marker forapoptosis) staining. The results show that the dying cells are Annexin Vpositive (FIG. 9A), demonstrating that compound 401 is apoptotic tocancer stem cells.

Alternatively, we performed CD133 (one of the common cancer stem cellsurface markers) antibody magnetic bead pull downs to enrich cancer stemcells. The CD133⁺ cells were then treated with compound 401 followed bystaining with antibody against cleaved-Caspase 3 (a hallmark ofapoptosis). As shown in FIG. 9B, many of the CD133⁺ cells becomecleaved-Caspase 3 positive after compound 401 treatment, corroboratingthat compound 401 is apoptotic to cancer stem cells.

In addition, we tested the activities of several other compounds againstcancer stem cells. Briefly, freshly isolated CSCs (SW480 Hoechst SPcells or CD44^(high) FaDu cells) were exposed to a dose range (30-0.117μM) of compound for 48 h before examining cell viability by MTT assay.IC₅₀s were estimated by plotting the percentage of surviving cells. Asshown in Table 4 and Table 5, compounds of present invention can targetcancer stem cells.

TABLE 4 IC₅₀ (μM) Compound NSP SP 401 0.33 0.34 418 0.33 0.34 4011 0.340.38 4012 0.81 0.57

TABLE 5 IC₅₀ (μM) Compound CD44^(low) CD44^(high) 4011 0.42 0.27 40120.76 1.05

Identification of Compounds that Inhibit CSC Spherogenesis In Vitro.

One of the hallmarks cancer stem cells is their ability to self-renew[87]. A reliable method of measuring the self-renewal capacity of cellpopulations is the ability to be cultured as spheres in the absence ofserum or attachment [88]. To compare the ability of compound 401 toother targeted and chemotherapeutic agents, FACS-isolated CD44^(high)CSCs were grown as spheres for 72 hours before being challenged with apanel of therapeutic agents. Of the agents tested, only compound 401 waseffective at preventing sphere proliferation (FIGS. 10A and 10B). Notethat spheres were resistant to doxorubicin and docetaxel despite beingapplied at approximately ten times their IC₅₀ concentrations for celldeath in similar assays. Tarceva, Sutent, and Gleevec were added atapproximately three times their reported therapeutic concentrations.This demonstrates that while cancer stem cells are resistant toconventional chemotherapeutic and targeted agents, compound 401 ishighly effective at inhibiting their growth.

Example 4

Identification of Compounds that Selectively Kill a Broad Spectrum ofCancer Cells

Identification of Compounds that are Apoptotic to a Broad Spectrum ofCancer Cells In Vitro.

Cells plated in 96 well plates and treated with indicated compounds weresubjected to MTT analysis at 24 hours following compound treatment todetermine cell viability. IC₅₀ values calculated across multiple celllines are summarized in Table 6 below. The data demonstrate that thesecompounds have potent activity against broad spectrum of cancer cells.

TABLE 6 Cell IC₅₀ (μM) Line Tissue 4011 4012 101 301 401 416 418 A549Lung 0.95 1.90 1.06 H1299 Lung 3-10 0.794 0.23 0.25 0.34 MCF7 Breast0.46 0.75 0.46 HeLa Cervix 11.7 3.358 0.43 0.62 0.80 DLD1 Colon 0.330.54 0.64 SW480 Colon 0.32 0.44 0.76 HCT116 Colon 0.58 0.69 0.61 HT29Colon 1.27 1.91 1.83 HepG2 Liver 0.25 Paca2 Pancreas 0.446 0.11 0.210.21 Panc1 Pancreas 1.70 2.59 1.54 DU145 Prostate  3.7 0.835 0.12 0.220.18 PC3 Prostate 2.37 3.10 3.04 LNCap Prostate 0.63 FaDu Head 0.3531.041 0.39 and Neck

Additionally, DU145 cells were first treated with DMSO or the indicatedconcentrations of compound 401, then stained with Annexin V, andfollowed by Flow cytometry analysis 5 hours post-treatment. The resultsshow that 401 treatment results in a dose-dependent increase in theAnnexin V staining (FIG. 11), demonstrating that compound 401 isapoptotic to these cancer cells.

Examples of the Selectivity of the Compounds.

Under assay conditions that result in the death of virtually all cancercells, normal cells remain substantially viable. Peripheral bloodmononuclear cells (PBMCs) were relatively resistant to compound 401,having an MTT IC₅₀ of 14 μM following a 24 hour incubation with thecompound 401. This IC₅₀ is between 6 to 116-fold greater than those seenin a variety of cancer cell lines, indicating a reasonable therapeuticwindow compared to cancer cells.

In a similar fashion to PMBCs, CD34⁺ bone marrow hematopoietic stemcells were spared when treated with compound 401. As shown in Table 7,incubation of CD34⁺ bone marrow mononuclear cells for 6 hours withcompound 401 resulted in an IC₅₀ of greater than 30 μM for both the bonemarrow erythroid and myeloid lineages, while DU145 prostate and HT29colon cancer cells have IC₅₀'s less than 0.5 μM under similarconditions. These data suggest a wide (greater than 50 fold) therapeuticwindow for compound 401 according to the in vitro data.

TABLE 7 Compound 401 is Relatively Non-Toxic to CD34⁺ Bone Marrow StemCells Compound 401 IC₅₀ (μM) Normal Cells Cancer Cells CD34⁺ BMErythroid CD34⁺ BM Myeloid DU145 HT29 >30 >30 <0.2 <0.5 BM = Bone MarrowComparison of the IC₅₀ of compound 401 on CD34⁺ Bone Marrow mononuclearcells, DU145 cells and FaDu cells in colony formation assays.

Example 8

Preparation of 2-Acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione (compound offormula 4-6 wherein R1═R7═H and R3═—CH₃)

To a solution of 18.8 gram (22 ml, 0.268 moles) of 3-buten-2-one(Formula 4-1 in scheme 1) in 200 ml of pentane in ice bath withvigorously stirring, was slowly added 39.0 grams (12.54 ml, 0.244 moles)of bromine in 50 ml of pentane within 30 minutes. After being stirredfor additional 5 minutes in ice bath, the mixture was evaporated toremove most of pentane. The small volume of 3,4-dibromo-2-butanoneresidue (4-2) from step 1 was dissolved in 400 ml of THF, and thenchilled in an ice bath. To the solution in ice bath with vigorouslystirring, was slowly added 37.2 grams (36.5 ml, 0.244 moles) of DBU in50 ml of THF within 30 minutes. Large quantity of precipitate salt wasgenerated. The mixture was directly used for next step reaction. To thereaction mixture of 3-bromo-3-buten-2-one (4-3), 38.5 grams (0.220moles) of 2-hydroxy-1,4-naphthoquinone (4-4) was added. The resultingmixture was stirred vigorously in a room temperature water bath. Then44.6 grams (43.8 ml, 0.293 moles) of DBU was slowly added to the mixturewithin 30 minutes. The temperature of the reaction mixture rose by theheat generated from reaction and was controlled to below 35° C. byadding ice to the water bath. After being vigorously stirred foradditional 3 hours in open air at room temperature, 1,800 ml of waterwas added to the mixture. The resulting mixture was chilled to 0° C. andthen filtered. The filtered solid was washed successively with 500 ml ofwater, 500 ml of 5% aqueous sodium bicarbonate, 500 ml of 1% acetic acidand 250 ml of ice chilled acetone. The washed solid was recrystallizedin 200 ml of formic acid to yield 12 grams of product with 22.8% overallyield for Compound 4-6 or 3-2 (R1═H, R3═CH₃, R7═H) ¹H NMR (in CDCl₃) δ2.67 (s, 3H), 7.61 (s, 1H-3), 7.79-7.84 (m, 2H), 8.22-8.28 (m, 2H).

Example 9 Scheme 2 Preparation of compound phosphoric acidmono-[1-(4,9-dioxo-3a,4,9,9a-tetrahydro-naphtho[2,3-b]furan-2-yl)-vinyl]ester

To a solution of BBI6002 (240 mg, 1 mmol) in 50 THF at −78° C. was addedlithium bis(trimethylsilyl)amide solution (1.0M in THF, 1.2 mL). Afterbeing stirred at this temperature for 1 hour, the reaction mixture wasstirred at 0° C. for 30 min. A solution of dimethyl chlorophosphate (217mg, 1.5 mmol) in 5 mL THF was then added to this mixture at −78° C. Theresulted mixture was stirred and allowed to warm to room temperatureslowly. After 1 h of stirring at ambient temperature the solvent wasevaporated, the residue was dissolved in CH₂Cl₂, washed with saturatedNH₄Cl and water and dried over MgSO₄. Evaporation of solvent affordedthe crude reaction mixture, which was purified from columnchromatography to yield phosphoric acid1-(4,9-dioxo-3a,4,9,9a-tetrahydro-naphtho[2,3-b]furan-2-yl)-vinyl esterdimethyl ester as yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 8.20-8.28 (m,2H), 7.78-7.82 (m, 2H), 7.07 (s, 1H), 5.78 (t, 1H, J=2.8 Hz), 5.50 (t,1H, J=2.8 Hz), 3.96 (s, 3H), 3.94 (s, 3H); MS m/z 347.20 (M-H).

Example 10 Scheme 3 Preparation of compound phosphoric acid1-(4,9-dioxo-3a,4,9,9a-tetrahydro-naphtho[2.3-b]furan-2-yl)-vinyl esterdimethyl ester

To a solution of phosphoric acid1-(4,9-dioxo-3a,4,9,9a-tetrahydro-naphtho[2,3-b]furan-2-yl)-vinyl esterdimethyl ester (40 mg, 0.114 mmol) in CH₂Cl₂ (2 mL) at room temperaturewas added trimethylsilyl bromide (71 mg, 0.46 mmol). The reactionmixture was stirred at room temperature for 3 hours and thenconcentrated in vacuum. The residue was purified by semi-prep-HPLC toobtain the product phosphoric acidmono-[1-(4,9-dioxo-3a,4,9,9a-tetrahydro-naphtho[2,3-b]furan-2-yl)-vinyl]ester as yellow solid. ¹H NMR (400 MHz, CDCl₃) δ ¹H NMR (400 MHz, CDCl₃)δ 7.98-8.02 (m, 2H), 7.57-7.62 (m, 2H), 6.92 (s, 1H), 5.54 (t, 1H, J=2.8Hz), 5.36 (t, 1H, J=2.8 Hz); MS m/z 319.20 (M-H).

All references cited herein are incorporated herein by reference intheir entirety to the extent allowed by applicable laws and for allpurposes to the same extent as if each individual publication or patentor patent application is specifically and individually indicated to beincorporated by reference in its entirety for all purposes. To theextent publications and patents or patent applications incorporated byreference contradict the disclosure contained in the specification, thespecification is intended to supersede and/or take precedence over anysuch contradictory material.

All numbers expressing quantities of ingredients, reaction conditions,analytical results and so forth used in the specification and claims areto be understood as being modified in all instances by the term “about.”Accordingly, unless indicated to the contrary, the numerical parametersset forth in the specification and attached claims are approximationsthat may vary depending upon the desired properties sought to beobtained by the present invention. At the very least, and not as anattempt to limit the application of the doctrine of equivalents to thescope of the claims, each numerical parameter should be construed inlight of the number of significant digits and ordinary roundingapproaches.

Modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only and are not meant to be limiting in anyway. It is intended that the specification and examples be considered asexemplary only, with a true scope and spirit of the invention beingindicated by the following claims.

REFERENCES

-   1. Bonnet, D., Normal and leukaemic stem cells. Br J Haematol, 2005.    130(4): p. 469-79.-   2. Bonnet, D. and J. E. Dick, Human acute myeloid leukemia is    organized as a hierarchy that originates from a primitive    hematopoietic cell. Nat Med, 1997. 3(7): p. 730-7.-   3. Hambardzumyan, D., M. Squatrito, and E. C. Holland, Radiation    resistance and stem-like cells in brain tumors. Cancer Cell, 2006.    10(6): p. 454-6.-   4. Baumann, M., M. Krause, and R. Hill, Exploring the role of cancer    stem cells in radioresistance. Nat Rev Cancer, 2008. 8(7): p.    545-54.-   5. Ailles, L. E. and I. L. Weissman, Cancer stem cells in solid    tumors. Curr Opin Biotechnol, 2007. 18(5): p. 460-6.-   6. Jones, R. J., W. H. Matsui, and B. D. Smith, Cancer stem cells:    are we missing the target? J Natl Cancer Inst, 2004. 96(8): p.    583-5.-   7. Ho, M. M., et al., Side population in human lung cancer cell    lines and tumors is enriched with stem-like cancer cells. Cancer    Res, 2007. 67(10): p. 4827-33.-   8. Wang, J., et al., Identification of cancer stem cell-like side    population cells in human nasopharyngeal carcinoma cell line. Cancer    Res, 2007. 67(8): p. 3716-24.-   9. Haraguchi, N., et al., Characterization of a side population of    cancer cells from human gastrointestinal system. Stem Cells, 2006.    24(3): p. 506-13.-   10. Doyle, L. A. and D. D. Ross, Multidrug resistance mediated by    the breast cancer resistance protein BCRP (ABCG2). Oncogene, 2003.    22(47): p. 7340-58.-   11. Alvi, A. J., et al., Functional and molecular characterisation    of mammary side population cells. Breast Cancer Res, 2003. 5(1): p.    R1-8.-   12. Frank, N. Y., et al., ABCB5-mediated doxorubicin transport and    chemoresistance in human malignant melanoma. Cancer Res, 2005.    65(10): p. 4320-33.-   13. Schatton, T., et al., Identification of cells initiating human    melanomas. Nature, 2008. 451(7176): p. 345-9.-   14. Kondo, T., T. Setoguchi, and T. Taga, Persistence of a small    subpopulation of cancer stem-like cells in the C6 glioma cell line.    Proc Natl Acad Sci USA, 2004. 101(3): p. 781-6.-   15. Goodell, M. A., et al., Isolation and functional properties of    murine hematopoietic stem cells that are replicating in vivo. J Exp    Med, 1996. 183(4): p. 1797-806.-   16. Collins, A. T., et al., Prospective identification of    tumorigenicprostate cancer stem cells. Cancer Res, 2005. 65(23): p.    10946-51.-   17. Li, C., et al., Identification of pancreatic cancer stem cells.    Cancer Res, 2007. 67(3): p. 1030-7.-   18. Ma, S., et al., Identification and characterization of    tumorigenic liver cancer stem/progenitor cells.    Gastroenterology, 2007. 132(7): p. 2542-56.-   19. Prince, M. E., et al., Identification of a subpopulation of    cells with cancer stem cell properties in head and neck squamous    cell carcinoma. Proc Natl Acad Sci USA, 2007. 104(3): p. 973-8.-   20. Ricci-Vitiani, L., et al., Identification and expansion of human    colon-cancer-initiating cells. Nature, 2007. 445(7123): p. 111-5.-   21. Singh, S. K., et al., Identification of a cancer stem cell in    human brain tumors. Cancer Res, 2003. 63(18): p. 5821-8.-   22. Dalerba, P., et al., Phenotypic characterization of human    colorectal cancer stem cells. Proc Natl Acad Sci USA, 2007.    104(24): p. 10158-63.-   23. Yu, H. Stat3: Linking oncogenesis with tumor immune evasion, in    AACR 2008 Annual Meeting. 2008, San Diego, Calif.-   24. Pedranzini, L., A. Leitch, and J. Bromberg, Stat3 is required    for the development of skin cancer. J Clin Invest, 2004. 114(5): p.    619-22.-   25. Catlett-Falcone, R., et al., Constitutive activation of Stat3    signaling confers resistance to apoptosis in human U266 myeloma    cells. Immunity, 1999. 10(1): p. 105-15.-   26. Bromberg, J. F., et al., Stat3 as an oncogene. Cell, 1999.    98(3): p. 295-303.-   27. Kanda, N., et al., STAT3 is constitutively activated and    supports cell survival in association with survivin expression in    gastric cancer cells. Oncogene, 2004. 23(28): p. 4921-9.-   28. Schlette, E. J., et al., Survivin expression predicts poorer    prognosis in anaplastic large-cell lymphoma. J Clin Oncol, 2004.    22(9): p. 1682-8.-   29. Niu, G., et al., Constitutive Stat3 activity up-regulates VEGF    expression and tumor angiogenesis. Oncogene, 2002, 21(13): p.    2000-8.-   30. Xie, T. X., et al., Stat3 activation regulates the expression of    matrix metalloproteinase-2 and tumor invasion and metastasis.    Oncogene, 2004. 23(20): p. 3550-60.-   31. Kortylewski, M., et al., Inhibiting Stat3 signaling in the    hematopoietic system elicits multicomponent antitumor immunity. Nat    Med, 2005, 11(12): p. 1314-21.-   32. Burdelya, L., et al., Stat 3 activity in melanoma cells affects    migration of immune effector cells and nitric oxide-mediated    antitumor effects. J Immunol, 2005. 174(7): p. 3925-31.-   33. Wang, T., et al., Regulation of the innate and adaptive immune    responses by Stat-3 signaling in tumor cells. Nat Med, 2004.    10(1): p. 48-54.-   34. Darnell, J. E., Validating Stat3 in cancer therapy. Nat    Med, 2005. 11(6): p. 595-6.-   35. Zhang, L., et al., Intratumoral delivery and suppression of    prostate tumor growth by attenuated Salmonella enterica serovar    typhimurium carrying plasmid-based small interfering RNAs. Cancer    Res, 2007. 67(12): p. 5859-64.-   36. Campbell, I. L., Cytokine-mediated inflammation, tumorigenesis,    and disease-associated JAK/STAT/SOCS signaling circuits in the CNS.    Brain Res Brain Res Rev, 2005. 48(2): p. 166-77.-   37. Harris, T. J., et al., Cutting edge: An in vivo requirement for    STAT3 signaling in TH17 development and TH17-dependent autoimmunity.    J Immunol, 2007. 179(7): p. 4313-7.-   38. Libby, P., P. M. Ridker, and A. Maseri, Inflammation and    atherosclerosis. Circulation, 2002. 105(9): p. 1135-43.-   39. Stephens, J. W., et al., A common functional variant in the    interleukin-6 gene is associated with increased body mass index in    subjects with type 2 diabetes mellitus. Mol Genet Metab, 2004.    82(2): p. 180-6.-   40. Cesari, M., et al., Inflammatory markers and onset of    cardiovascular events: results from the Health ABC study.    Circulation, 2003. 108(19): p. 2317-22.-   41. Orshal, J. M. and R. A. Khalil, Interleukin-6 impairs    endothelium-dependent NO-cGMP-mediated relaxation and enhances    contraction in systemic vessels of pregnant rats. Am J Physiol Regul    Integr Comp Physiol, 2004. 286(6): p. R1013-23.-   42. Manolagas, S. C., Role of cytokines in bone resorption.    Bone, 1995. 17(2 Suppl): p. 63S-67S.-   43. Yaffe, K., et al., Inflammatory markers and cognition in    well-functioning African-American and white elders. Neurology, 2003.    61(1): p. 76-80.-   44. Watson, C. J. and W. R. Miller, Elevated levels of members of    the STAT family of transcription factors in breast carcinoma nuclear    extracts. Br J Cancer, 1995. 71(4): p. 840-4.-   45. Song, J. I. and J. R. Grandis, STAT signaling in head and neck    cancer. Oncogene, 2000. 19(21): p. 2489-95.-   46. Song, L., et al., Activation of Stat3 by receptor tyrosine    kinases and cytokines regulates survival in human non-small cell    carcinoma cells. Oncogene, 2003. 22(27): p. 4150-65.-   47. Savarese, T. M., et al., Coexpression of oncostatin M and its    receptors and evidence for STAT3 activation in human ovarian    carcinomas. Cytokine, 2002. 17(6): p. 324-34.-   48. Toyonaga, T., et al., Blockade of constitutively activated Janus    kinase/signal transducer and activator of transcription-3 pathway    inhibits growth of human pancreatic cancer. Cancer Lett, 2003.    201(1): p. 107-16.-   49. Corvinus, F. M., et al., Persistent STAT3 activation in colon    cancer is associated with enhanced cell proliferation and tumor    growth. Neoplasia, 2005. 7(6): p. 545-55.-   50. Gao, B., et al., Constitutive activation of JAK-STAT3 signaling    by BRCA1 in human prostate cancer cells. FEBS Lett, 2001. 488(3): p.    179-84.-   51. Buettner, R., L. B. Mora, and R. Jove, Activated STAT signaling    in human tumors provides novel molecular targets for therapeutic    intervention. Clin Cancer Res, 2002. 8(4): p. 945-54.-   52. Carson, W. E., Interferon-alpha-induced activation of signal    transducer and activator of transcription proteins in malignant    melanoma. Clin Cancer Res, 1998. 4(9): p. 2219-28.-   53. Chen, C. L., et al., Stat3 activation in human endometrial and    cervical cancers. Br J Cancer, 2007. 96(4): p. 591-9.-   54. Lai, R., et al., STAT3 is activated in a subset of the Ewing    sarcoma family of tumours. J Pathol, 2006. 208(5): p. 624-32.-   55. Punjabi, A. S., et al., Persistent activation of STAT3 by latent    Kaposi's sarcoma-associated herpesvirus infection of endothelial    cells. J Virol, 2007. 81(5): p. 2449-58.-   56. Schaefer, L. K., et al., Constitutive activation of Stat3alpha    in brain tumors: localization to tumor endothelial cells and    activation by the endothelial tyrosine kinase receptor (VEGFR-2).    Oncogene, 2002. 21(13): p. 2058-65.-   57. Puthier, D., R. Bataille, and M. Amiot, IL-6 up-regulates mcl-1    in human myeloma cells through JAK/STAT rather than ras/MAP kinase    pathway. Eur J Immunol, 1999. 29(12): p. 3945-50.-   58. Migone, T. S., et al., Constitutively activated Jak-STAT pathway    in T cells transformed with HTLV-I. Science, 1995. 269(5220): p.    79-81.-   59. Spiekermann, K., et al., Constitutive activation of STAT    transcription factors in acute myelogenous leukemia. Eur J    Haematol, 2001. 67(2): p. 63-71.-   60. Epling-Burnette, P. K., et al., Inhibition of STAT3 signaling    leads to apoptosis of leukemic large granular lymphocytes and    decreased Mcl-1 expression. J Clin Invest, 2001. 107(3): p. 351-62.-   61. Weber-Nordt, R. M., et al., Constitutive activation of STAT    proteins in primary lymphoid and myeloid leukemia cells and in    Epstein-Barr virus (EBV)-related lymphoma cell lines. Blood, 1996.    88(3): p. 809-16.-   62. Sommer, V. H., et al., In vivo activation of STAT3 in cutaneous    T-cell lymphoma. Evidence for an antiapoptotic function of STAT3.    Leukemia, 2004. 18(7): p. 1288-95.-   63. Lai, R., et al., Signal transducer and activator of    transcription-3 activation contributes to high tissue inhibitor of    metalloproteinase-1 expression in anaplastic lymphoma    kinase-positive anaplastic large cell lymphoma. Am J Pathol, 2004.    164(6): p. 2251-8.-   64. Fu, X. Y., STAT3 in immune responses and inflammatory bowel    diseases. Cell Res, 2006. 16(2): p. 214-9.-   65. Feldmann, M., F. M. Brennan, and R. N. Maini, Role of cytokines    in rheumatoid arthritis. Annu Rev Immunol, 1996. 14: p. 397-440.-   66. Krause, A., et al., Rheumatoid arthritis synoviocyte survival is    dependent on Stat3. J Immunol, 2002. 169(11); p. 6610-6.-   67. Pfitzner, E., et al., The role of STATs in inflammation and    inflammatory diseases. Curr Pharm Des, 2004. 10(23): p. 2839-50.-   68. Lovato, P., et al., Constitutive STAT3 activation in intestinal    T cells from patients with Crohn's disease. J Biol Chem, 2003.    278(19): p. 16777-81.-   69. Ishihara, K. and T. Hirano, IL-6 in autoimmune disease and    chronic inflammatory proliferative disease. Cytokine Growth Factor    Rev, 2002. 13(4-5): p. 357-68.-   70. Ivashkiv, L. B. and I. Tassiulas, Can SOCS make arthritis    better? J Clin Invest, 2003. 111(6): p. 795-7.-   71. Sengupta, T. K., et al., Activation of monocyte effector genes    and STAT family transcription factors by inflammatory synovial fluid    is independent of interferon gamma. J Exp Med, 1995. 181(3): p.    1015-25.-   72. Shouda, T., et al., Induction of the cytokine signal regulator    SOCS3/CIS3 as a therapeutic strategy for treating inflammatory    arthritis. J Clin Invest, 2001. 108(12): p. 1781-8.-   73. Harada, T., et al., Increased expression of STAT3 in SLE T cells    contributes to enhanced chemokine-mediated cell migration.    Autoimmunity, 2007. 40(1): p. 1-8.-   74. Simeone-Penney, M. C., et al., Airway epithelial STAT3 is    required for allergic inflammation in a murine model of asthma. J    Immunol, 2007. 178(10): p. 6191-9.-   75. Hagler, M., Smith-Norowitz, T., Chice, S., Wallner, S., Viterbo,    D., Mueller, C., Groos, R., Nowakowski, M., Schulze, R., Zenilman,    M., Sophorolipids decrease IgE production in U266 cells by    downregulation of BSAP (Pax5), TLR-2, STAT3 and IL-6. Journal of    Allergy and Clinical Immunology, 2007. 119(S1): p. S263-S263.-   76. Benkhart, E. M., et al., Role of Stat3 in    lipopolysaccharide-induced IL-10 gene expression. J Immunol, 2000.    165(3): p. 1612-7.-   77. Sano, S., et al., Stat3 links activated keratinocytes and    immunocytes required for development of psoriasis in a novel    transgenic mouse model. Nat Med, 2005. 11(1): p. 43-9.-   78. Lim, C. P., et al., Stat3 contributes to keloid pathogenesis via    promoting collagen production, cell proliferation and migration.    Oncogene, 2006. 25(39): p. 5416-25.-   79. Arany, I., et al., Correlation between pretreatment levels of    interferon response genes and clinical responses to an immune    response modifier (Imiquimod) in genital warts. Antimicrob Agents    Chemother, 2000. 44(7): p. 1869-73.-   80. Tefferi, A., Classification, diagnosis and management of    myeloproliferative disorders in the JAK2V617F era. Hematology Am Soc    Hematol Educ Program, 2006: p. 240-5.-   81. Roder, S., et al., STAT3 is constitutively active in some    patients with Polycythemia rubra vera. Exp Hematol, 2001. 29(6): p.    694-702.-   82. Kim, O. S., et al., JAK-STAT signaling mediates    gangliosides-induced inflammatory responses in brain microglial    cells. J Biol Chem, 2002. 277(43): p. 40594-601.-   83. Wyss-Coray, T., Inflammation in Alzheimer disease: driving    force, bystander or beneficial response? Nat Med, 2006. 12(9): p.    1005-15.-   84. Stelmasiak, Z., et al., Interleukin-6 concentration in serum and    cerebrospinal fluid in multiple sclerosis patients. Med Sci    Monit, 2000. 6(6): p. 1104-8.-   85. Ponti, D., et al., Isolation and in vitro propagation of    tumorigenic breast cancer cells with stem/progenitor cell    properties. Cancer Res, 2005. 65(13): p. 5506-11.-   86. Szotek, P. P., et al., Ovarian cancer side population defines    cells with stem cell-like characteristics and Mullerian Inhibiting    Substance responsiveness. Proc Natl Acad Sci USA, 2006. 103(30): p.    11154-9.-   87. Al-Hajj, M., et al., Therapeutic implications of cancer stem    cells. Curr Opin Genet Dev, 2004. 14(1): p. 43-7.-   88. Bleau, A. M., et al., New strategy for the analysis of    phenotypic marker antigens in brain tumor-derived neurospheres in    mice and humans. Neurosurg Focus, 2008. 24(3-4): p. E28.

1. A method of inhibiting a cancer stem cell, the method comprisinginhibiting at least some Stat3 pathway activity in the cancer stem cellthrough a Stat3 pathway inhibitor.